EP2214705A2 - A powerful vaccine composition comprising a lipopeptide and poly i:c as an adjuvant - Google Patents

A powerful vaccine composition comprising a lipopeptide and poly i:c as an adjuvant

Info

Publication number
EP2214705A2
EP2214705A2 EP08856104A EP08856104A EP2214705A2 EP 2214705 A2 EP2214705 A2 EP 2214705A2 EP 08856104 A EP08856104 A EP 08856104A EP 08856104 A EP08856104 A EP 08856104A EP 2214705 A2 EP2214705 A2 EP 2214705A2
Authority
EP
European Patent Office
Prior art keywords
antigen
cancer
adjuvant
skkkk
vaccine
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
EP08856104A
Other languages
German (de)
French (fr)
Other versions
EP2214705B1 (en
EP2214705A4 (en
Inventor
Hong Mo Moon
Byung Cheol Ahn
Jung Sun Yum
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Dobeel Co Ltd
Original Assignee
Dobeel Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Dobeel Co Ltd filed Critical Dobeel Co Ltd
Publication of EP2214705A2 publication Critical patent/EP2214705A2/en
Publication of EP2214705A4 publication Critical patent/EP2214705A4/en
Application granted granted Critical
Publication of EP2214705B1 publication Critical patent/EP2214705B1/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/385—Haptens or antigens, bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/29—Hepatitis virus
    • A61K39/292—Serum hepatitis virus, hepatitis B virus, e.g. Australia antigen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A61P33/06—Antimalarials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
    • C07K16/082—Hepadnaviridae, e.g. hepatitis B virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to an adjuvant comprising a lipopeptide and poly I:C (polyinosinic:polycytidylic acid) and a vaccine comprising the same.
  • An adjuvant plays a role in promoting immune response by accelerating or amplifying one or more specific phases of various immune response inducing processes. Therefore, when an adjuvant is co-administered with an antigen, it can improve immunogenicity of the antigen and/or alters the type of immune response against the antigen.
  • Typical examples of such adjuvant are oil emulsion (Freund's adjuvant), monophosphoryl lipid A (MPL), Q saponins, aluminum hydroxide or phosphate or calcium salts (alum) of aluminum, non-ionic block polymer surfactants, lipopolysaccharides, mycobacteria, tetanus toxoid, CpG, etc.
  • the vaccine composition contains normally antigen and an adjuvant.
  • a signal delivered through the engagement of antigen epitope presented with MHC molecule and antigen receptor is not enough to induce an immune response. It requires additional signals generated from costimulatory molecule(s).
  • the adjuvant may be able to enforce signal strength generated by costimulatory molecules and/or induce costimulatory molecules and also induce cytokines that determine the type of immune responses.
  • Some antigens such as lipoproteins, glycoproteins, or whole microorganisms can provide both epitopes and adjuvant function in the form of pathogen associated molecular pattern (PAMP).
  • PAMP pathogen associated molecular pattern
  • the primary structure of protein antigens that is, the amino acid sequence of an antigen cannot be changed, but the PAMP of an antigen can be modified or supplemented by the addition of a proper adjuvant or subsidiary structure to affect immunogenicity (Dempsey PW et al, Science 111 : 348-350, 1996; Deres K et al, Nature 342: 561-564, 1989).
  • the modification of a molecular pattern of an antigen can increase immunogenicity and also affect the type of elicited immune response.
  • pathogen recognition receptor existing on the surface of antigen presenting cell(APC) recognizes PAMP to generate signals to induce various costimulatory molecules and cytokines, which affects the type of immune response as well as the magnitude.
  • PAMP pathogen recognition receptor
  • interferon gamma and IL- 12 helps to induce ThI (T helper cell 1) response playing an important role in immune response against vims infection.
  • ThI type immune response leads to the increase of IgG2a and IgG2b generation and induce powerful cell mediated immune response.
  • various types of PAMPs associated with antigens are playing as an adjuvant functions and such adjuvant can help in regulating immune responses.
  • these types of natural adjuvant function associated with the antigens may not be strong enough to induce desired strength and quality of immune responses, requiring a good adjuvant in vaccine formulation.
  • TLR Toll Like Receptors
  • APC antigen presenting cells
  • PLC antigen presenting cells
  • PLC antigen presenting cells
  • Pam3cys which is one of TLR2 ligands, works in inducing immune response independently of TLR2 (Yoder et al, Infect. Immun. 71:3894-3900, 2003).
  • good protective immune response requires balanced immune response comprising both strong cell mediated immune response and humoral antibody response. Therefore, developing an adjuvant that will help to induce well balanced adaptive immune response is still beyond the matter that can be rationally predicted.
  • inventors have defined a powerful vaccine as a vaccine formulation that can generate a large amount of high quality antigen specific antibody in reference to the most well known adjuvant aluminum hydroxide.
  • the generation of an appropriate, high quality antibody is a very important factor for producing a good preventive or an effective therapeutic vaccine.
  • IgG isotypes play different roles in elimination of a tumor cells; IgG2a is the most effective one, compared with IgGl, IgG2b or IgG3 (Nimmerjahn F & Ravetch JV 5 Science 310: 1510-1512, 2005).
  • IgG2a and IgG2b known to be the most effective in inducing antiviral immunity (Coutelier JP et al, J Exp Med 165:64-69, 1987; Markine-Gorianoff D & Coutelier JP, J of Virol 76:432-435, 2002), are generated by cytokines produced by ThI cells, which also induce cell mediated immune response. Therefore, the induction of ThI cell response is a good indication for the generation of an appropriate, high quality antibody.
  • the most widely utilized adjuvant, Alum induces Th 2 type immune response, and induced antibody is mainly IgGl.
  • the powerful vaccine composition of the present invention is judged by the amount of an antigen specific antibody generated and high ratios of IgG2a/IgGl and IgG2b/IgGl comparing to widely utilized Alum adjuvant containing vaccine.
  • the adjuvant described in this invention is the one that is able to induce powerful antibody response as well as cell-mediated immune response and that can switch immunoglobulin isotype to produce IgG2a and IgG2b.
  • an adjuvant composition comprising lipopeptide and poly LC (polyinosinic:polycytidylic acid) is far more powerful than the conventional adjuvant, aluminum hydroxide, and that further confirming lipopeptide and Poly LC are synergistic instead of additive in stimulation of adaptive immune responses.
  • Pam3Cys and poly LC is known to be synergistic in inducing TNF- ⁇ and IL-6 in macrophage (Bagchi,A.et al, J. Immun. 178:1164-1171, 2007)
  • well balanced powerful adjuvant function of a similar combination consisting of a lipopeptide and poly LC is an unexected finding.
  • Present inventors confirmed further that covalent linking of lipopeptide to antigen is not required. Simple formulation in the form of a mixture of a lipopeptide, poly LC, and at least one antigen is sufficient.
  • the present invention provides an adjuvant comprising a lipopeptide and poly I:C (polyinosinic:polytidylic acid).
  • the present invention also provides a vaccine composition containing the above adjuvant and at least one apropriate antigen.
  • the present invention also provides a method for generating an appropriate, high quality antibody comprising steps of administrating the above vaccine composition to a subject in need thereof.
  • the present invention also provides a method for enhancing ThI immune response comprising steps of administrating the above vaccine composition to a subject in need thereof.
  • the present invention also provides a therapeutic vaccine against viral or parasite infection, comprising the adjuvant composition and at least one viral or parasite antigen.
  • the present invention also provides a prophylactic or therapeutic vaccine against cancer, containing the adjuvant composition and at least one cancer-specific antigen.
  • the present invention also provides use of the adjuvant and at least one viral or parasite antigen in the manufacture of a therapeutic agent for treating viral or parasite infection.
  • the present invention provides use of the adjuvant and at least one cancer-specific antigen in the manufacture of a therapeutic agent for treating cancer.
  • the adjuvant of the present invention When the adjuvant of the present invention is used together with an antigen, the antigen specific antibody induction is stimulated and ThI type immune response is also induced. Therefore, the adjuvant of the present invention is very effective as an adjuvant for vaccine formulation for the prevention and treatment of viral or parasite infection or cancer.
  • Figure 1 is a graph showing the titer of S-protein antibody elicited by various vaccine formulations containing L-HBsAg composed of L-protein (S-protein-preS2-preSl), M-protein (S-protein-preS2), and S-protein in aluminum hydroxide (Alum), Pam3Cys-SKKKK alone, poly I:C alone, or both Pam3Cys-SKKKK and poy I:C as adjuvants.
  • L-HBsAg composed of L-protein (S-protein-preS2-preSl), M-protein (S-protein-preS2), and S-protein in aluminum hydroxide (Alum), Pam3Cys-SKKKK alone, poly I:C alone, or both Pam3Cys-SKKKK and poy I:C as adjuvants.
  • Figure 2 is a graph showing the titer of antibody against preS antigen induced by various vaccine formulations with L-HBsAg and Alum, Pam3Cys-SKKKK alone, poly I:C alone, or both Pam3Cys-SKKKK and Poly I:C as adjuvants. In this experiment pronounced effect of synergy between Pam3Cys-SKKKK and poly I:C can be seen.
  • Figure 3 is a set of graphs showing the antibody isotypes induced by various different vaccine formulations with L-HBsAg in Alum, Pam3Cys-SKKKK alone, poly I:C alone, or both Pam3Cys-SKKKK and Poly I:C as adjuvants, a; Antibody titer of each isotype, b; IgG2a/IgGl, c; IgG2b/IgGl.
  • Figure 4 is a graph showing the immunogenicity against Influenza virus HA antigen formulated with different adjuvants; Alum, Pam3Cys-SKKKK alone, poly I:C alone, or both Pam3Cys-SKKKK and Poly LC.
  • Figure 5 is a set of graphs showing the antibody isotypes induced by Influenza virus antigen formulated with different types of adjuvants; Alum, Pam3Cys-SKKKK alone, poly LC alone, or both Pam3Cys-SKKKK and Poly LC. a; Antibody titer of each isotype b; IgG2a/IgGl
  • Figure 6 is a graph showing the immunogenicity against HBsAg antigen (S-protein) from Hansenula polymorpha and preS from Saccharomyces cereviciae formulated in different adjuvants; Alum, Pam3Cys-SKKKK alone, poly LC alone, or both Pam3Cys-SKKKK.
  • Figure 7 is a graph showing the immunogenicity against preS antigen by vaccines formulated with Hansenula S-protein and preS from yeast in different adjuvants; Alum, Pam3Cys-SKKKK alone, poly LC alone, or both Pam3Cys-SKKKK and Poly LC.
  • Figure 8 is a set of graphs showing the antibody isotypes induced by vaccines formulated with HBsAg (S-protein) from Hansenula and preS from yeast in different adjuvants; Alum, Pam3Cys-SKKKK alone, poly LC alone or both Pam3Cys-SKKKK.
  • FIG. 9 is a graph showing the titer of S-protein antibody elicited by various vaccine formulations containing L-HBsAg composed of L-protein (S-protein-preS2-preSl), M-protein (S-protein-preS2), and S-protein in aluminum hydroxide (Alum), Pam3Cys-SKKKK alone, Pam3Cys-SR8 alone, FSL-I alone, poly LC alone, combination of Pam3Cys-SKKKK and poy LC, combination of Pam3Cys-SR8 and poy LC, or combination of FSL-I and poy LC as adjuvants.
  • L-HBsAg composed of L-protein (S-protein-preS2-preSl), M-protein (S-protein-preS2), and S-protein in aluminum hydroxide (Alum)
  • Figure 10 is a graph showing the titer of antibody against preS antigen induced by various vaccine formulations with L-HBsAg and aluminum hydroxide (Alum),
  • Pam3Cys-SKKKK and poy LC combination of Pam3Cys-SR8 and poy LC, or combination of FSL-I and poy LC as adjuvants.
  • Figure 11 is a set of graphs showing the antibody isotypes induced by various different vaccine formulations with L-HBsAg in aluminum hydroxide (Alum), Pam3Cys-SKKKK alone, Pam3Cys-SR8 alone, FSL-I alone, poly LC alone, combination of
  • FSL-I and poy LC as adjuvants.
  • the present invention provides an adjuvant for vaccine, comprising one or more lipopeptides and poly LC (polyinosinic:polytidylic acid).
  • poly LC polyinosinic:polytidylic acid
  • Pam3Cys-SKKKK, a kind of lipopeptides, and poly I: C were mixed and this mixture was used as an adjuvant to produce a vaccine with L-HBsAg 5 influenza antigen, or a mixture of HBsAg S-protein and PreS as antigens.
  • IgGl antibody was predominant IgG isotype.
  • a vaccine was formulated using the mixture comprising Pam3Cys-SKKKK and poly LC, IgG2a and IgG2b were produced dominantly. Therefore, the ratios of IgG2a/IgGl and IgG2b/IgGl were higher with the mixture of Pam3Cys-SKKKK and Poly LC compare to the ratio obtained with conventional adjuvant aluminum hydroxide (see Figures 3, 5 and 8, and Tables I 9 2 and 3).
  • the lipopeptide was first synthesized by Metzger et al. as a synthetic analogue of lipopeptide originated from bacteria and mycoplasma (Metzger J et al. , Int J Peptide Protein Res 37:46-57, 1991). Since then, numbers of analogues have been synthesized (EMC microcollections GmbH Sindelfinger Str. 372070 Tubingen, Germany). There is a report that virus-specific cytotoxic T lymphocyte (CTL) was induced by administrating a mouse with Pani3Cys-Ser-Ser, a kind of lipopeptides conjugated with influenza virus T cell epitope (Schild H et al, Eur J Immunol 21:2649-2654, 1991). In general, lipopeptide has been known as a TLR2 ligand (Trinchieri G & Sher A, Nat Rev Immunol 7: 179-190, 2007).
  • CTL virus-specific cytotoxic T lymphocyte
  • the lipopeptide is composed of fatty acids linked to glycerol and amino acids. Lipopeptides contain one or more fatty acids in each molecule.
  • the lipopeptide can be a lipoprotein composed of a part of or a whole molecule originated from gram positive or gram negative bacteria or mycoplasma. And, the fatty acid and amino acid herein can be synthesized with chemical modifications.
  • the lipopeptide herein is exemplified by Pam3Cys-SKKKK (formula 1; molecular structure: N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)- ⁇ ropyl]-[R]-cystein-SKKKK), PHC-SKKKK, Ole2PamCys-SKKKK, Pam2Cys-SKKKK, PamCys(Pam)-SKKKK, Ole2Cys-SKKKK, Myr2Cys-SKKKK, PamDhc-SKKKK, PamCSKKKK and Dhc-SKKKK, but not always limited thereto.
  • Poly I:C has been used as a powerful inducer of type I interferon in in vitro and in vivo studies (Magee ME & Griffith MJ, life Science II, 11 :1081-1086, 1972; Manetti YR et al, Eur J Immunol 25:2656-2660, 1995), and has been known to induce dendritic cell (DC) maturation, most popular antigen presenting cell (APC) in mammals.
  • DC dendritic cell
  • APC antigen presenting cell
  • Poly I:C is also known as an IL- 12 inducer, and the IL- 12 is an important cytokine inducing cell mediated immune response and IgG2a antibody generation by promoting the enhancement of ThI development. Adjuvant activity of poly I:C was also shown previously (Cui Z & Qui F, Cancer Immunol Immunotherapy 16:1-13, 2005).
  • the poly I:C (polyinosinic:porycytidylic acid) is a synthetic double stranded RNA, and the length is preferably 50 - 2000 bp and more preferably 100 - 500 bp.
  • the present invention provides a vaccine composition containing said adjuvant comprising a lipopeptide and poly I:C and at least one antigen. More specifically present invention provides an adjuvant comprising one or more lipopeptides and poly LC that can stimulate immune response synergistically instead of giving additive effect by each adjuvant component.
  • a vaccine prepared by using the adjuvant of the present invention was proved to increase synergistically antigen specific antibody production as well as changing the quality of immune response, inducing mostly IgG2a and IgG2b (see Figures 1 - 8 and Tables 1 - 3). Therefore, an adjuvant composition containing said adjuvant components of the present invention can be effectively used to increase immunogenicity of antigen and thereby improving the efficacy of vaccine.
  • the antigen herein indicates any material that can induce immune response by the immune system of animal or human. It can be a full length or a fragment
  • the antigen can be provided as synthetic material, purified subunits, a whole microbe or a mixture, but purified antigen is preferred.
  • the antigen herein is exemplified by a recombinant protein, or peptide from hepatitis virus or viral protein from influenza virus, but it can be a polysaccharide, a glycoprotein, a lipopolysaccharide, a DNA molecule, a cancer cell, a live virus or a hapten molecule, etc.
  • influenza virus antigen haemagglutinin or neuraminidase antigen
  • HA haemagglutinin or neuraminidase antigen
  • it can be Bordetella pertussis antigen, pertussis toxin, filamentous haemagglutinin, human papilloma virus (HPV) antigen, Helicobacter pylori antigen (capsular polysaccharides of serogrup A, B, C, Y and W- 135), tetanus toxoid, diphtheria antigen (diphtheria toxoid), pneumococcal antigen ⁇ Streptococcus pnemoniae type 3 capsular polysaccharides), tuberculosis antigen, human immunodeficiency virus (HIV) antigen (GP-120, GP-160), cholera antigen (cholera toxin B subunit), staphylococcal antigen (staphylococcal enterotoxin B
  • the vaccine composition of the present invention can additionally include, in addition to the adjuvant and an antigen, one or more effective ingredients having the same or similar effect with them.
  • the vaccine composition can also include, in addition to the above-mentioned effective ingredients, one or more pharmaceutically acceptable carriers for the administration.
  • the pharmaceutically acceptable carrier can be selected or be prepared by mixing more than one ingredients selected from a group consisting of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrose solution, glycerol and ethanol. Other general additives such as anti-oxidative agent, buffer solution, bacteriostatic agent, etc., can be added.
  • the vaccine composition of the present invention can further be prepared in suitable forms for each disease or according to ingredients by following a method represented in Remington's Pharmaceutical Science (the newest edition, Mack Publishing Company, Easton, PA).
  • the vaccine composition of the present invention can be administered parenterally by various routs such as subcutaneous injection, intravenous injection, intramuscular injection and intrathoracic injection.
  • the vaccine composition is mixed with a stabilizer or a buffering agent to produce a solution or suspension, which is then formulated as a content of ampoules, syringes, or vials in single or multiple doses.
  • the vaccine composition of the present invention can be formulated in various forms according to the administration pathway.
  • the vaccine composition can be prepared as a sterilized aqueous solution or suspension for injection or a freeze-dried form.
  • the freeze-dried vaccine composition is stored typically at 4 ° C and can be restored with a stabilizer containing with or without an additive such as saline and/or HEPES.
  • the dosage of the vaccine composition of the present invention can be determined by considering administration method and frequency, or disease under treatment, severity of disease, history of disease, co-treatment with another therapeutic agent or not, and age, height, weight, health condition, or physical condition, but not always limited thereto. In general, the dose of this vaccine composition is preferably increased according to the weight increase of a patient under treatment.
  • the present invention also provides a method for generating an appropriate, high quality antibody comprising the step of administering the above vaccine composition to a subject.
  • the vaccine composition prepared by using the adjuvant of the present invention increased antigen specific antibody production and IgG2a and IgG2b production (see Figures 1 — 10 and Tables 1 - 4). Therefore, the vaccine composition of the present invention can be effectively used for the mass-production of an appropriate, high quality antibody in which stimulation of immunogenicity of antigen is required.
  • the vaccine composition can be administered parenterally, by intraperitoneal injection, hypodermic injection, intravenous injection or intramuscular injection.
  • the vaccine composition can be administered at the dose enough to stimulate immune response in a subject.
  • the vaccine can be administered to human once or several times, each time at the dose of 1 - 250 ⁇ g and more preferably 10 - 100 ⁇ g.
  • the present invention also provides a method for enhancing ThI immune response, comprising procedures of administering the above vaccine composition to a subject in need thereof.
  • the vaccine composition prepared by using the adjuvant of the present invention increased IgG2a and IgG2b production (see
  • the vaccine composition of the present invention can be effectively used for enhancing ThI immune response to improve immunogenicity
  • the present invention also provides an immune therapeutic agent for viral or parasite infection, containing the said adjuvant and at least one viral or parasite antigen composition as active ingredients.
  • the present invention also provides use of the adjuvant and at least one viral or parasite antigen in the manufacture of a therapeutic agent for treating viral ro parasite infection.
  • the therapeutic agent composition prepared by using the adjuvant of the present invention increased antigen specific antibody production and IgG2a and IgG2b production (see Figures 1 - 10 and Tables 1 - 4).
  • IgG2a and IgG2b are known to be effective in defending viral infection and cellular infection of parasites. Therefore, the vaccine composition comprising the adjuvant for vaccine of the present invention and at least one viral or parisite antigen can be effectively used as a therapeutic agent for viral or parasite infection.
  • the viral antigen is influenza virus antigen (HA: haemagglutinin or neuraminidase antigen), human papilloma virus (HPV) antigen, human immunodeficiency virus (HIV) antigen (GP- 120, GP- 160), vesicular stomatitis virus antigen (vesicular stomatitis virus glycoprotein), cytomegalovirus (CMV) antigen, hepatitis antigen [hepatitis A(HAV), B(HBV), C(HCV), D(HDV) and G(HGV): L-HBsAg, S-HBsAg, M-HBsAg, pre S], respiratory synctytial virus (RSV) antigen or herpes simplex virus antigen.
  • HA haemagglutinin or neuraminidase antigen
  • HPV human papilloma virus
  • HAV human immunodeficiency virus
  • HIV vesicular stomatitis
  • the parasite is protozoa, nematoda, trematoda or cestoda but not limited thereto.
  • the protozoa is preferrably a rhizopoda, a mastigophora, a ciliate or a sporozoa but not limited thereto.
  • the rhizopoda includes Entamoeba histoytica and Entamoeba coli.
  • the mastigophora includes Giardia lamblia, Trichomonas vaginalis, Trichomonas hominis and Haemoflagellates.
  • the cilliate includes Balantidium coli.
  • the sporozoa is preferrably a Plasmodium sp. including P. vivax and P. falciparum, a Toxoplasma gondii, Pneumocystis carinii, Isospora hominis, Cryptosporidium sp. including C. parvum and C. muris.
  • the nematoda is preferrably a whipworm, a hookworm, a pinworm, an ascarid or a filariodea but not limited thereto.
  • the whipworm is preferrably Trichuris trichiura or Trichocephalus trichiuris but not limited thereto. However, any other whipworms infecting animals such as dogs, cats and pigs may be included.
  • the hookworm is preferrably Ancylostoma duodenale or Necator americanus but not limited thereto.
  • the pinworm is preferrably Enterobius vermicularis or Enterobius gregorii.
  • the ascarid is preferrably A. suum which typically infects pigs or A. lumbricoides which infects humans.
  • the filariodea is preferrably Wuchereria bancrofti, Onchocerca volvulus, Loa loa or Dirofilaria immitis.
  • the trematoda is preferably Digenea, but not limited thereto.
  • the Digenea includes Schistosome or non-Schistosome.
  • the schistosome includes Schistosoma mansoni, Schistosoma haematobium, Schistosoma japonicun and Schistosoma intercalatum, but not limited thereto.
  • the non-Schistosome includes Fasciolopsis buski, Heterophyes heterophyes, Metagonimus yokogawaii, Gastrodiscoides hominis, Clonorchis sinensis, Fasciola hepatica and Paragonimus westermani, but not limited thereto.
  • the cestoda is preferably Taeniidae or Diphyllobothriidae but not limited thereto.
  • the Taeniidae includes Taenia solium and Taenia saginata.
  • the Diphyllobothriidae includes Diphyllobothrium sp. such as Diphyllobothrium latum, Diphyllobothrium dendriticum, D. nihonkaiense, D. pacificum, D. cordatum, D. ursi, D. lanceolatum, D. dalliae, and D. yonagoensis.
  • the "parasite antigen” means a molecule derived from a parasite which is capable of inducing humoral response in a host.
  • the parasite antigen can be a surface glyco- protein or a carbohydrate molecule thereof or a lipid molecule.
  • WO9526402A1 discloses a helminth parasite antigen characterized by
  • WO951267 IAl discloses a helminth parasite antigen possesing aminopeptidase-like activity.
  • US6399077 discloses a noninfectious soluble fraction of a Toxoplasma gondii infected cell culture lysate.
  • US6413521 discloses an isolated and purified antigen conferring protective immunity against a non-obligate blood feeding helminth and which is characterized by possessing aminopeptidase M-like activity.
  • US20070087012A1 discloses a novel Fasciclin Related Adhesive Protein (FRAP) from Plasmodium and related parasites.
  • FRAP Fasciclin Related Adhesive Protein
  • WO9402169A1 discloses a protective metazoan parasite antigen capable of binding to pepstatin.
  • US4656251 discloses parasite antigens of Dirofilaria immitis.
  • the therapeutic agent against viral or parasite infection can be administered parenterally by hypodermic injection, intravenous injection or intramuscular injection.
  • the vaccine composition of the present invention is prepared as an oil emulsion, which is then formulated as ampoules, syringes or vials.
  • the effective dosage can be determined according to absorption rate, inactivation rate, age, gender, health condition of a patient, and severity of disease, etc.
  • the present invention also provides a preventive or therapeutic agent for cancer containing the adjuvant and at least one cancer-specific antigen composition as active ingredients.
  • the cancer is preferably a renal cell carcinoma, a melanoma, a chronic lymphocytic leukemia, a lung cancer, a cervical cancer, a stomach cancer, a thyroid cancer, a pancreatic cancer, a breast cancer, a prostate cancer, an ovarian cancer, a cholangioma, a liver cancer, a colon cancer, or a lectal cancer, but not limited thereto.
  • cancer-specific antigen is a ptotein or an immulogically active fragment thereof which is differentially expressed in cancerous tissues rather than normal tissues.
  • Various cancer-specific antigens are known in the art. For example, gplOO, MART-I and MAGE-I are well-known antigen specific for menanoma.
  • the other cancer-specific antigen includes tyrosinase, CEA (cancer embryonic antigen), PSA (prostate specific antigen), HER2/neu, MAGE-I, MAGE-2, MAGE-3, NY-ESO-I, MUC-I, SART-I or SART-3, TERT (telomerase reverse transcriptase) or partial peptides derived from TERT, WTl or partial peptides derived from WTl, Survivin-2B or partial peptides derived from Survivin-2B, gp75, MDM2, telomerase, alph-1 fetoprotein, CA125, CA15-3, CA19-9, G250 and NY-ESO-I 5 but not limited thereto (See WO 2006/078059 and WO 2007/065957).
  • CAA cancer-associated antigen
  • the present invention provides use of the adjuvant as a vaccine composition in the manufacture of an immunological therapeutic agent for treating cancer, in that strong cellular immune response is required.
  • the immunological therapeutic agent composition prepared by using the adjuvant of the present invention increased antigen-specific antibody production and IgG2a and IgG2b production (see Figures 1 — 10 and Tables 1 - 4). IgG2a and IgG2b are known to be very effective in anticancer immune response. Therefore, the immunological therapeutic agent composition containing the adjuvant for vaccine of the present invention and an appropriate cancer-specific antigen can be effectively used as a preventive or therapeutic agent for preventing or treating cancer.
  • the immunological agent for preventing or treating cancer can be administered parenterally by hypodermic injection, intravenous injection or intramuscular injection.
  • the vaccine composition of the present invention is formulated as an oil emulsion, which is then stored as ampoules, syringes, or vials.
  • the effective dosage can be determined according to absorption rate, inactivation rate, age, gender, health condition of a patient, and severity of disease, etc.
  • Example 1 Stimulation of immunogenicity of hepatitis B virus (HBV) antigen
  • Vaccines were prepared with hepatitis B virus antigen and various adjuvants including aluminum hydroxide (Alum; Brenntag Biosector, Germany), Pam3Cys-SKKKK (lipopeptide) (EMC microcollections GmbH, Germany) alone, poly I:C (Sigma, USA) alone, or the mixture of both Pam3Cys-SKKKK and poly I:C, and the induced antibody titer of each vaccine formulation was compared.
  • Vaccines were prepared by mixing hepatitis B virus whole surface antigen (L-HBsAg;
  • L-HBsAg is consisted of S-protein (small protein without pre S 1 and pre S2), M-protein (medium protein with pre S2 only), and L-protein (large protein with both preSl and preS2).
  • Serum was collected before the vaccine administration and 2 weeks after the third vaccine administration and antigen specific antibody generation was analyzed by ELISA to determine antibody titer.
  • a 96-well microplate was coated with recombinant S-protein (Dobeel Corp., Korea) at the concentration of 100 ng/well and blocked by adding 1% BSA for one hour.
  • the microplate was washed and apropriately diluted serum was added to each well and incubated at 37 ° C for 2 hours.
  • Anti mouse goat IgG-HRP Horse Radish Peroxidase; Sigma, USA
  • Antibody titer was determined by the same way as described in Example ⁇ l-2-l> except that preS antigen (Dobeel Corp., Korea) was used as an antibody capturing antigen. As shown in Table 1 and Figure 2, the adjuvant mixture containing both
  • Pam3Cys-SKKKK and poly I:C was more effective in inducing higher antibody titer against preS.
  • Induction of pre S antibody by the adjuvant mixture was synergistic, inducing more than 4 times of pre S antibody compare to the added value of pre S antibody titer induced by Pam3Cys-SKKKK alone and poly I:C alone.
  • Antibody titer was determined by the same manner as described in Example ⁇ l-2-l> except IgGl, IgG2a and IgG2b (mouse monoclonal antibody isotyping reagents; Sigma, USA) were used as a secondary antibody. And IgG2a/IgGl and IgG2b/IgGl ratios were also calculated by using the obtained antibody titer.
  • isotypes IgG2a and IgG2b were much higher with the adjuvant mixture comparing to the values obtained with aluminum hydroxide.
  • the induction of IgG2b was more than 20 times of the value obtained with aluminum hydroxide.
  • IgG2a/IgGl ratio was about 10 times higher than aluminmum hydroxide.
  • the production of IgG2b was significantly high, and IgG2b/IgGl ratio was much higher than IgG2a/IgGl ( Figure 3b and Figure 3c).
  • Example 2 Stimulation of immunogenicity of influenza virus antigen
  • Influenza virus antigen was formulated with aluminum hydroxide, Pam3Cys-SKKKK alone, poly I:C alone, or with the mixture of Pam3Cys-SKKKK and Poly I:C as adjuvants and induced antibody titer was determined using influenza virus antigen as capturing antigen as for HBsAg antibody assay.
  • Different formulations were prepared by mixing recombinant split vaccine antigen (Korea Vaccine Co., Ltd, Korea) and the said adjuvant, which were administered to mice.
  • the antigen was prepared by infecting the allantoic sac of a developing egg with influenza virus strains A/New Caledonia/20/99(H1N1), a/Wisconsin/67/2005(H3N2) and B/Malaysia/2506/2004, culturing, purifying and inactivating thereof.
  • Serum was collected from each mouse before the vaccine administration and 2 weeks after the second vaccine administration and antigen specific antibody generation was analyzed by ELISA to determine antibody titer.
  • Antibody titer was determined by the same manner as described in Example ⁇ l-2-l> except that HA antigen (Korea Vaccine Co., Ltd, Korea) was used as an antibody capturing antigen.
  • Antibody titer was determined by the same way as described in Example ⁇ 2-2-l> except that IgGl and IgG2a were used as secondary antibodies for isotype determination. IgG2a/IgGl and IgG2b/IgGl ratios were also calculated by using the obtained antibody titer.
  • Figure 5 a shows antibody titers of isotypes IgGl, and IgG2a. Isotype ratios were also calculated using the antibody titers. IgG2a/IgGl value was specifically high ( Figure 5b) with the adjuvant mixture compare to the value obtained with single components or Aluminum hydroxide.
  • Example 3 Preparation of a powerful vaccine using recombinant HBsAg (S-protein from Hansenula polymorph ⁇ ) and recombinant preS protein (from Saccharomyces cerevisiae)
  • Vaccines were prepared by mixing recombinant HBsAg (Dobeel Corp., Korea), recombinant preS protein (Dobeel Corp., Korea) and said adjuvants, and they were administered by intra-muscular injection to mice.
  • the recombinant HBsAg contained only S-protein without preS antigen and the recombinant preS protein prepared as a particle type by conjugating them to colloidal gold were used as antigens.
  • a mixture containing 20 ⁇ g each of Pam3Cys-SKKKK and poly I:C was used as adjuvant with 0.5 ⁇ g of recombinant S-protein and 5 ⁇ g of preS protein to give a vaccine in oil emulsion form.
  • Each test vaccine contained 0.5 ⁇ g of the recombinant S-protein and 5 ⁇ g of the preS per dose, which was then given by intramuscular injection to 5 week old C57BL/6 female mice three times at two weeks intervals.
  • Negative control is the group received PBS only. Positive control is the group received the mixture comprising aluminum hydroxide, S-protein, and colloidal gold conjugated recombinant preS antigen.
  • Experimental group 1 is the group received vaccine prepared by mixing emulsified S-protein, Pam3Cys-SKKKK and poly I:C and colloidal gold conjugated recombinant preS antigen.
  • Experimental group 2 is the group received vaccine prepared by emulsification of all components together, including S-protein, colloidal gold conjugated recombinant preS antigen and the mixture of Pam3Cys-SKKKK and poly LC.
  • Antibody titer against S-protein was determined by the same way as described in Example ⁇ l-2-l>.
  • the adjuvant mixture of Pam3Cys-SKKKK and poly LC induced more than 10 times higher antibody titer against S-protein than aluminum hydroxide.
  • the vaccine prepared by emulsifying all components together induced higher antibody titer than the vaccine prepared by emulsifying S-protein with the said adjuvant first, and adding the conjugated pre S.
  • Pre S antibody titer was determined by the same manner as described in Example ⁇ l-2-2>.
  • Isotype antibody titer was determined by the same method as described in Example ⁇ l-2-3>. And IgG2a/IgGl and IgG2b/IgGl ratios were calculated from the obtained antibody titer.
  • antibody titers of isotypes IgGl, IgG2a and IgG2b were obtained. Isotype ratios were also calculated using the antibody titers.
  • the adjuvant mixture of Pam3Cys-SKKKK and poly I:C was used, IgG2a/IgGl and IgG2b/IgGl ratios were higher comparing to aluminum hydroxide.
  • the vaccine prepared by mixing HBsAg and preS antigen with the said adjuvant and emulsifying together, was confirmed more effective than the vaccine prepared by mixing preS antigen with emulsified HBsAg and the said adjuvant ( Figures 8b and 8c).
  • Vaccines were prepared by mixing hepatitis B virus whole surface antigen that was preadsorbed on aluminum hydroxide and said adjuvant components, and they were administered to mice. In the course of experiments, it was noticed that the aluminum hydroxide adsorbed antigen is more stable, and the aluminum hydroxide has no noticeable effect on the adjuvant effect of lipopeptide and poly LC mixture (data not presented).
  • Vaccines were injected intra muscularly into 6 weeks old C57BL/6 female mice three times at two weeks intervals.
  • the negative control was administered only with PBS without the vaccine and the antigen, while the positive control was administered with the said antigen formulated with aluminum hydroxide.
  • Antibody titer against HBsAg (S-protein) Antibody titer was determined by the same method as described in Example ⁇ l-2-l>.
  • Antibody titer was determined by the same way as described in Example ⁇ l-2-l> except that preS antigen (Dobeel Corp., Korea) was used as an antibody capturing antigen.
  • the adjuvant mixture containing one of the lipopeptides and poly LC was more effective in inducing higher antibody titer against preS.
  • Antibody titer was determined by the same manner as described in Example ⁇ l-2-l> except IgGl 5 IgG2a and IgG2b were used as a secondary antibody. And IgG2a/IgGl and IgG2b/IgGl ratios were also calculated by using the obtained antibody titer.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Virology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Epidemiology (AREA)
  • Mycology (AREA)
  • Microbiology (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pulmonology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Hematology (AREA)
  • AIDS & HIV (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The present invention relates to an adjuvant comprising a lipopeptide and poly I:C. When the adjuvant comprising a lipopeptide and poly I:C of the present invention is used, the level of antigen specific antibody induction is synergistically increased and ThI type immune response is also induced. Therefore, the adjuvant of the present invention can be very effectively used as an adjuvant in the formulation of preventive and therapeutic vaccines for viral infection and cancer.

Description

DESCRIPTION Invention Title
A POWERFUL VACCINE COMPOSITION COMPRISING A LIPOPEPTIDE AND POLY I:C AS AN ADJUVANT
Technical Field
The present invention relates to an adjuvant comprising a lipopeptide and poly I:C (polyinosinic:polycytidylic acid) and a vaccine comprising the same. Background Art
An adjuvant plays a role in promoting immune response by accelerating or amplifying one or more specific phases of various immune response inducing processes. Therefore, when an adjuvant is co-administered with an antigen, it can improve immunogenicity of the antigen and/or alters the type of immune response against the antigen. Typical examples of such adjuvant are oil emulsion (Freund's adjuvant), monophosphoryl lipid A (MPL), Q saponins, aluminum hydroxide or phosphate or calcium salts (alum) of aluminum, non-ionic block polymer surfactants, lipopolysaccharides, mycobacteria, tetanus toxoid, CpG, etc.
Lfee of a protein antigen alone does not often induce strong enough immune response and a desired type of immune response, so the vaccine composition contains normally antigen and an adjuvant. According to two signal model for immune responses, a signal delivered through the engagement of antigen epitope presented with MHC molecule and antigen receptor is not enough to induce an immune response. It requires additional signals generated from costimulatory molecule(s). In this the adjuvant may be able to enforce signal strength generated by costimulatory molecules and/or induce costimulatory molecules and also induce cytokines that determine the type of immune responses. Some antigens such as lipoproteins, glycoproteins, or whole microorganisms can provide both epitopes and adjuvant function in the form of pathogen associated molecular pattern (PAMP). The primary structure of protein antigens, that is, the amino acid sequence of an antigen cannot be changed, but the PAMP of an antigen can be modified or supplemented by the addition of a proper adjuvant or subsidiary structure to affect immunogenicity (Dempsey PW et al, Science 111 : 348-350, 1996; Deres K et al, Nature 342: 561-564, 1989). " The modification of a molecular pattern of an antigen can increase immunogenicity and also affect the type of elicited immune response. For example, in the case of HBV surface antigen, S-protein without preS 1 and preS2 does not exhibit immunogenicity in certain congenic mouse strains, while L-protein containing preSl and preS2 not only induces antibody generation against preSl and preS2 but also helps to induce antibody generation against S antigen (Milich DR et al, 1986. New Approaches to Immunization, pp 377-382. Cold Spring Harbor Laboratories, New York). In the case when a whole pathogenic microorganism is used as an antigen, it is expected for the microorganism to contain various types of PAMP such as lipopolysaccharides, nucleic acids, lipoproteins and conjugated proteins. In this situation, pathogen recognition receptor (PRR) existing on the surface of antigen presenting cell(APC) recognizes PAMP to generate signals to induce various costimulatory molecules and cytokines, which affects the type of immune response as well as the magnitude. For example, interferon gamma and IL- 12 helps to induce ThI (T helper cell 1) response playing an important role in immune response against vims infection. ThI type immune response leads to the increase of IgG2a and IgG2b generation and induce powerful cell mediated immune response. In this, various types of PAMPs associated with antigens are playing as an adjuvant functions and such adjuvant can help in regulating immune responses. However, often, these types of natural adjuvant function associated with the antigens may not be strong enough to induce desired strength and quality of immune responses, requiring a good adjuvant in vaccine formulation.
Therefore, developing a good adjuvant is a very important job in developing a good vaccine, but an adjuvant development still has to be relied mainly on empirical work. For example, Toll Like Receptors (TLR) are the most important PRR on antigen presenting cells (APC) involved in the activation of APC and in antigen presentation by APC. Potent antibody response, however, is not entirely dependent on TLR signals (Gavin. A. L. et al, Science 314:1936-1938, 2006). Further, Pam3cys, which is one of TLR2 ligands, works in inducing immune response independently of TLR2 (Yoder et al, Infect. Immun. 71:3894-3900, 2003). To make matters further complicated, good protective immune response requires balanced immune response comprising both strong cell mediated immune response and humoral antibody response. Therefore, developing an adjuvant that will help to induce well balanced adaptive immune response is still beyond the matter that can be rationally predicted.
In this invention, inventors have defined a powerful vaccine as a vaccine formulation that can generate a large amount of high quality antigen specific antibody in reference to the most well known adjuvant aluminum hydroxide. The generation of an appropriate, high quality antibody is a very important factor for producing a good preventive or an effective therapeutic vaccine. For example, IgG isotypes play different roles in elimination of a tumor cells; IgG2a is the most effective one, compared with IgGl, IgG2b or IgG3 (Nimmerjahn F & Ravetch JV5 Science 310: 1510-1512, 2005). IgG2a and IgG2b, known to be the most effective in inducing antiviral immunity (Coutelier JP et al, J Exp Med 165:64-69, 1987; Markine-Gorianoff D & Coutelier JP, J of Virol 76:432-435, 2002), are generated by cytokines produced by ThI cells, which also induce cell mediated immune response. Therefore, the induction of ThI cell response is a good indication for the generation of an appropriate, high quality antibody. The most widely utilized adjuvant, Alum, induces Th 2 type immune response, and induced antibody is mainly IgGl.
Thus, the powerful vaccine composition of the present invention is judged by the amount of an antigen specific antibody generated and high ratios of IgG2a/IgGl and IgG2b/IgGl comparing to widely utilized Alum adjuvant containing vaccine. The adjuvant described in this invention is the one that is able to induce powerful antibody response as well as cell-mediated immune response and that can switch immunoglobulin isotype to produce IgG2a and IgG2b.
Present inventors completed this invention by confirming that an adjuvant composition comprising lipopeptide and poly LC (polyinosinic:polycytidylic acid) is far more powerful than the conventional adjuvant, aluminum hydroxide, and that further confirming lipopeptide and Poly LC are synergistic instead of additive in stimulation of adaptive immune responses. Although Pam3Cys and poly LC is known to be synergistic in inducing TNF-α and IL-6 in macrophage (Bagchi,A.et al, J. Immun. 178:1164-1171, 2007), well balanced powerful adjuvant function of a similar combination consisting of a lipopeptide and poly LC is an unexected finding. Present inventors confirmed further that covalent linking of lipopeptide to antigen is not required. Simple formulation in the form of a mixture of a lipopeptide, poly LC, and at least one antigen is sufficient.
Disclosure
Technical objectives
It is an objective of the present invention to provide an adjuvant that can help antigen to induce strong immune response. It is another objective of the present invention to provide a vaccine composition containing the above adjuvant and an antigen.
It is further an objective of the present invention to provide a method for generating an appropriate, high quality antibody using the above adjuvant. It is also an objective of the present invention to provide a method for enhancing ThI immune response using the aforementioned adjuvant.
It is also an objective of the present invention to provide a therapeutic vaccine for viral or parasite infection, containing aforementioned adjuvant and at least one viral or parasite antigen. It is also an objective of the present invention to provide a preventive or therapeutic vaccine against cancer, containing the adjuvant and at least one cancer-specific antigen.
Technical Solution
To achieve above objectives, the present invention provides an adjuvant comprising a lipopeptide and poly I:C (polyinosinic:polytidylic acid).
The present invention also provides a vaccine composition containing the above adjuvant and at least one apropriate antigen.
The present invention also provides a method for generating an appropriate, high quality antibody comprising steps of administrating the above vaccine composition to a subject in need thereof.
The present invention also provides a method for enhancing ThI immune response comprising steps of administrating the above vaccine composition to a subject in need thereof. The present invention also provides a therapeutic vaccine against viral or parasite infection, comprising the adjuvant composition and at least one viral or parasite antigen. The present invention also provides a prophylactic or therapeutic vaccine against cancer, containing the adjuvant composition and at least one cancer-specific antigen.
The present invention also provides use of the adjuvant and at least one viral or parasite antigen in the manufacture of a therapeutic agent for treating viral or parasite infection. In addition, the present invention provides use of the adjuvant and at least one cancer-specific antigen in the manufacture of a therapeutic agent for treating cancer.
Advantageous Effect
When the adjuvant of the present invention is used together with an antigen, the antigen specific antibody induction is stimulated and ThI type immune response is also induced. Therefore, the adjuvant of the present invention is very effective as an adjuvant for vaccine formulation for the prevention and treatment of viral or parasite infection or cancer.
Description of Drawings The application of the preferred embodiments of the present invention is best understood with reference to the accompanying drawings, wherein:
Figure 1 is a graph showing the titer of S-protein antibody elicited by various vaccine formulations containing L-HBsAg composed of L-protein (S-protein-preS2-preSl), M-protein (S-protein-preS2), and S-protein in aluminum hydroxide (Alum), Pam3Cys-SKKKK alone, poly I:C alone, or both Pam3Cys-SKKKK and poy I:C as adjuvants.
Figure 2 is a graph showing the titer of antibody against preS antigen induced by various vaccine formulations with L-HBsAg and Alum, Pam3Cys-SKKKK alone, poly I:C alone, or both Pam3Cys-SKKKK and Poly I:C as adjuvants. In this experiment pronounced effect of synergy between Pam3Cys-SKKKK and poly I:C can be seen. Figure 3 is a set of graphs showing the antibody isotypes induced by various different vaccine formulations with L-HBsAg in Alum, Pam3Cys-SKKKK alone, poly I:C alone, or both Pam3Cys-SKKKK and Poly I:C as adjuvants, a; Antibody titer of each isotype, b; IgG2a/IgGl, c; IgG2b/IgGl.
Figure 4 is a graph showing the immunogenicity against Influenza virus HA antigen formulated with different adjuvants; Alum, Pam3Cys-SKKKK alone, poly I:C alone, or both Pam3Cys-SKKKK and Poly LC.
Figure 5 is a set of graphs showing the antibody isotypes induced by Influenza virus antigen formulated with different types of adjuvants; Alum, Pam3Cys-SKKKK alone, poly LC alone, or both Pam3Cys-SKKKK and Poly LC. a; Antibody titer of each isotype b; IgG2a/IgGl
Figure 6 is a graph showing the immunogenicity against HBsAg antigen (S-protein) from Hansenula polymorpha and preS from Saccharomyces cereviciae formulated in different adjuvants; Alum, Pam3Cys-SKKKK alone, poly LC alone, or both Pam3Cys-SKKKK.
Figure 7 is a graph showing the immunogenicity against preS antigen by vaccines formulated with Hansenula S-protein and preS from yeast in different adjuvants; Alum, Pam3Cys-SKKKK alone, poly LC alone, or both Pam3Cys-SKKKK and Poly LC. Figure 8 is a set of graphs showing the antibody isotypes induced by vaccines formulated with HBsAg (S-protein) from Hansenula and preS from yeast in different adjuvants; Alum, Pam3Cys-SKKKK alone, poly LC alone or both Pam3Cys-SKKKK. a; Antibody titer of each isotype, b; IgG2a/IgGl, c; IgG2b/IgGl. Figure 9 is a graph showing the titer of S-protein antibody elicited by various vaccine formulations containing L-HBsAg composed of L-protein (S-protein-preS2-preSl), M-protein (S-protein-preS2), and S-protein in aluminum hydroxide (Alum), Pam3Cys-SKKKK alone, Pam3Cys-SR8 alone, FSL-I alone, poly LC alone, combination of Pam3Cys-SKKKK and poy LC, combination of Pam3Cys-SR8 and poy LC, or combination of FSL-I and poy LC as adjuvants.
Figure 10 is a graph showing the titer of antibody against preS antigen induced by various vaccine formulations with L-HBsAg and aluminum hydroxide (Alum),
Pam3Cys-SKKKK alone, Pam3Cys-SR8 alone, FSL-I alone, poly LC alone, combination of
Pam3Cys-SKKKK and poy LC, combination of Pam3Cys-SR8 and poy LC, or combination of FSL-I and poy LC as adjuvants.
In this experiment pronounced effect of synergy between lipopeptide and poly LC can be seen.
Figure 11 is a set of graphs showing the antibody isotypes induced by various different vaccine formulations with L-HBsAg in aluminum hydroxide (Alum), Pam3Cys-SKKKK alone, Pam3Cys-SR8 alone, FSL-I alone, poly LC alone, combination of
Pam3Cys-SKKKK and poy LC, combination of Pam3Cys-SR8 and poy LC, or combination of
FSL-I and poy LC as adjuvants. a; Antibody titer of each isotype, b; IgG2a/IgGl, c; IgG2b/IgGl.
Best Mode
Hereinafter, the present invention is described in detail.
The present invention provides an adjuvant for vaccine, comprising one or more lipopeptides and poly LC (polyinosinic:polytidylic acid). In a preferred embodiment of the present invention, Pam3Cys-SKKKK, a kind of lipopeptides, and poly I: C were mixed and this mixture was used as an adjuvant to produce a vaccine with L-HBsAg5 influenza antigen, or a mixture of HBsAg S-protein and PreS as antigens. And the vaccine containing mixture of Pam3Cys-SKKKK and Poly I:C was confirmed to enhance the antigen-specific antibody production significantly, compared to most frequently used conventional adjuvant aluminum hydroxide (see Figures I9 2, 4, 6 and 7, and Tables 1, 2 and 3).
When the mixture of Pam3Cys-SKKKK and Poly I:C was used, it was synergistic in stimulating immune responses; that is, the titer of pre S antibody induced by the mixuture was several times more than the combined value of the antibody titer induced by individual components of the mixture. This is exemplified best in the figure 2, in which the antibody titer of pre S antibody was assessed for the mixture or individual components. This synergistic effect is less pronounced for S-protein antibody induction (Fig.l). This is due to the amount of antigen used in all expriments was saturating amount instead of right amount that will show adjuvant dependency. Pre S content in L-protein is less than 10% of the total (5 mg).
When a vaccine was formulated using aluminum hydroxide, which is known to induce Th2 immune response, IgGl antibody was predominant IgG isotype. Whereas, when a vaccine was formulated using the mixture comprising Pam3Cys-SKKKK and poly LC, IgG2a and IgG2b were produced dominantly. Therefore, the ratios of IgG2a/IgGl and IgG2b/IgGl were higher with the mixture of Pam3Cys-SKKKK and Poly LC compare to the ratio obtained with conventional adjuvant aluminum hydroxide (see Figures 3, 5 and 8, and Tables I9 2 and 3). This increase of IgG2a and IgG2b known to be very effective in defense against viral infection and cancer suggests the improvement of quality of immune response with the new adjuvant. This findings indicate that the adjuvant comprising lipopeptide and poly LC, the present invention, can be effectively used for the development of powerful therapeutic and prophylactic vaccine formulations.
The lipopeptide was first synthesized by Metzger et al. as a synthetic analogue of lipopeptide originated from bacteria and mycoplasma (Metzger J et al. , Int J Peptide Protein Res 37:46-57, 1991). Since then, numbers of analogues have been synthesized (EMC microcollections GmbH Sindelfinger Str. 372070 Tubingen, Germany). There is a report that virus-specific cytotoxic T lymphocyte (CTL) was induced by administrating a mouse with Pani3Cys-Ser-Ser, a kind of lipopeptides conjugated with influenza virus T cell epitope (Schild H et al, Eur J Immunol 21:2649-2654, 1991). In general, lipopeptide has been known as a TLR2 ligand (Trinchieri G & Sher A, Nat Rev Immunol 7: 179-190, 2007).
In this invention, the lipopeptide is composed of fatty acids linked to glycerol and amino acids. Lipopeptides contain one or more fatty acids in each molecule. The lipopeptide can be a lipoprotein composed of a part of or a whole molecule originated from gram positive or gram negative bacteria or mycoplasma. And, the fatty acid and amino acid herein can be synthesized with chemical modifications. The lipopeptide herein is exemplified by Pam3Cys-SKKKK (formula 1; molecular structure: N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-ρropyl]-[R]-cystein-SKKKK), PHC-SKKKK, Ole2PamCys-SKKKK, Pam2Cys-SKKKK, PamCys(Pam)-SKKKK, Ole2Cys-SKKKK, Myr2Cys-SKKKK, PamDhc-SKKKK, PamCSKKKK and Dhc-SKKKK, but not always limited thereto.
Formula 1
Poly I:C has been used as a powerful inducer of type I interferon in in vitro and in vivo studies (Magee ME & Griffith MJ, life Science II, 11 :1081-1086, 1972; Manetti YR et al, Eur J Immunol 25:2656-2660, 1995), and has been known to induce dendritic cell (DC) maturation, most popular antigen presenting cell (APC) in mammals. Mature DC is capable of inducding immune response effectively (Rous R et a!., International Immunol 16:767-773, 2004). Poly I:C is also known as an IL- 12 inducer, and the IL- 12 is an important cytokine inducing cell mediated immune response and IgG2a antibody generation by promoting the enhancement of ThI development. Adjuvant activity of poly I:C was also shown previously (Cui Z & Qui F, Cancer Immunol Immunotherapy 16:1-13, 2005). The poly I:C (polyinosinic:porycytidylic acid) is a synthetic double stranded RNA, and the length is preferably 50 - 2000 bp and more preferably 100 - 500 bp.
The present invention provides a vaccine composition containing said adjuvant comprising a lipopeptide and poly I:C and at least one antigen. More specifically present invention provides an adjuvant comprising one or more lipopeptides and poly LC that can stimulate immune response synergistically instead of giving additive effect by each adjuvant component.
In a preferred embodiment of the present invention, a vaccine prepared by using the adjuvant of the present invention was proved to increase synergistically antigen specific antibody production as well as changing the quality of immune response, inducing mostly IgG2a and IgG2b (see Figures 1 - 8 and Tables 1 - 3). Therefore, an adjuvant composition containing said adjuvant components of the present invention can be effectively used to increase immunogenicity of antigen and thereby improving the efficacy of vaccine. The antigen herein indicates any material that can induce immune response by the immune system of animal or human. It can be a full length or a fragment The antigen can be provided as synthetic material, purified subunits, a whole microbe or a mixture, but purified antigen is preferred.
The antigen herein is exemplified by a recombinant protein, or peptide from hepatitis virus or viral protein from influenza virus, but it can be a polysaccharide, a glycoprotein, a lipopolysaccharide, a DNA molecule, a cancer cell, a live virus or a hapten molecule, etc. The protein of a pathogen above is exemplified by influenza virus antigen (HA: haemagglutinin or neuraminidase antigen), but it can be Bordetella pertussis antigen, pertussis toxin, filamentous haemagglutinin, human papilloma virus (HPV) antigen, Helicobacter pylori antigen (capsular polysaccharides of serogrup A, B, C, Y and W- 135), tetanus toxoid, diphtheria antigen (diphtheria toxoid), pneumococcal antigen {Streptococcus pnemoniae type 3 capsular polysaccharides), tuberculosis antigen, human immunodeficiency virus (HIV) antigen (GP-120, GP-160), cholera antigen (cholera toxin B subunit), staphylococcal antigen (staphylococcal enterotoxin B), shigella antigen (shigella polysaccharides), vesicular stomatitis virus antigen (vesicular stomatitis virus glycoprotein), cytomegalovirus (CMV) antigen, hepatitis antigen [hepatitis A(HAV), B(HBV), C(HCV), D(HDV) and G(HGV): L-HBsAg, S-HBsAg, M-HBsAg, pre S], respiratory synctytial virus (RSV) antigen, herpes simplex antigen and combinations thereof (ex: diphtheria, pertussis and tetanus; DPT), but not always limited thereto. The vaccine composition of the present invention can additionally include, in addition to the adjuvant and an antigen, one or more effective ingredients having the same or similar effect with them. The vaccine composition can also include, in addition to the above-mentioned effective ingredients, one or more pharmaceutically acceptable carriers for the administration. The pharmaceutically acceptable carrier can be selected or be prepared by mixing more than one ingredients selected from a group consisting of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrose solution, glycerol and ethanol. Other general additives such as anti-oxidative agent, buffer solution, bacteriostatic agent, etc., can be added. In order to prepare injectable solutions such as aqueous solution, suspension and emulsion, diluents, dispersing agents, surfactants, binders and lubricants can be additionally added. The vaccine composition of the present invention can further be prepared in suitable forms for each disease or according to ingredients by following a method represented in Remington's Pharmaceutical Science (the newest edition, Mack Publishing Company, Easton, PA).
The vaccine composition of the present invention can be administered parenterally by various routs such as subcutaneous injection, intravenous injection, intramuscular injection and intrathoracic injection. To prepare the vaccine composition as a formulation for parenteral administration, the vaccine composition is mixed with a stabilizer or a buffering agent to produce a solution or suspension, which is then formulated as a content of ampoules, syringes, or vials in single or multiple doses. The vaccine composition of the present invention can be formulated in various forms according to the administration pathway. For example, the vaccine composition can be prepared as a sterilized aqueous solution or suspension for injection or a freeze-dried form. The freeze-dried vaccine composition is stored typically at 4°C and can be restored with a stabilizer containing with or without an additive such as saline and/or HEPES. The dosage of the vaccine composition of the present invention can be determined by considering administration method and frequency, or disease under treatment, severity of disease, history of disease, co-treatment with another therapeutic agent or not, and age, height, weight, health condition, or physical condition, but not always limited thereto. In general, the dose of this vaccine composition is preferably increased according to the weight increase of a patient under treatment.
The present invention also provides a method for generating an appropriate, high quality antibody comprising the step of administering the above vaccine composition to a subject. In a preferred embodiment of the present invention, the vaccine composition prepared by using the adjuvant of the present invention increased antigen specific antibody production and IgG2a and IgG2b production (see Figures 1 — 10 and Tables 1 - 4). Therefore, the vaccine composition of the present invention can be effectively used for the mass-production of an appropriate, high quality antibody in which stimulation of immunogenicity of antigen is required.
In a preferred embodiment of the present invention, the vaccine composition can be administered parenterally, by intraperitoneal injection, hypodermic injection, intravenous injection or intramuscular injection. The vaccine composition can be administered at the dose enough to stimulate immune response in a subject. For example, the vaccine can be administered to human once or several times, each time at the dose of 1 - 250 μg and more preferably 10 - 100 μg.
The present invention also provides a method for enhancing ThI immune response, comprising procedures of administering the above vaccine composition to a subject in need thereof.
In a preferred embodiment of the present invention, the vaccine composition prepared by using the adjuvant of the present invention increased IgG2a and IgG2b production (see
Figures 3, 5, 8 and 10, and Tables 1, 2, 3 and 4). Therefore, the vaccine composition of the present invention can be effectively used for enhancing ThI immune response to improve immunogenicity
The present invention also provides an immune therapeutic agent for viral or parasite infection, containing the said adjuvant and at least one viral or parasite antigen composition as active ingredients. The present invention also provides use of the adjuvant and at least one viral or parasite antigen in the manufacture of a therapeutic agent for treating viral ro parasite infection.
In a preferred embodiment of the present invention, the therapeutic agent composition prepared by using the adjuvant of the present invention increased antigen specific antibody production and IgG2a and IgG2b production (see Figures 1 - 10 and Tables 1 - 4). IgG2a and IgG2b are known to be effective in defending viral infection and cellular infection of parasites. Therefore, the vaccine composition comprising the adjuvant for vaccine of the present invention and at least one viral or parisite antigen can be effectively used as a therapeutic agent for viral or parasite infection. In a preferred embodiment of the present invention, the viral antigen is influenza virus antigen (HA: haemagglutinin or neuraminidase antigen), human papilloma virus (HPV) antigen, human immunodeficiency virus (HIV) antigen (GP- 120, GP- 160), vesicular stomatitis virus antigen (vesicular stomatitis virus glycoprotein), cytomegalovirus (CMV) antigen, hepatitis antigen [hepatitis A(HAV), B(HBV), C(HCV), D(HDV) and G(HGV): L-HBsAg, S-HBsAg, M-HBsAg, pre S], respiratory synctytial virus (RSV) antigen or herpes simplex virus antigen.
In preferred embodiment of the present invention, the parasite is protozoa, nematoda, trematoda or cestoda but not limited thereto.
The protozoa is preferrably a rhizopoda, a mastigophora, a ciliate or a sporozoa but not limited thereto.
The rhizopoda includes Entamoeba histoytica and Entamoeba coli. The mastigophora includes Giardia lamblia, Trichomonas vaginalis, Trichomonas hominis and Haemoflagellates.
The cilliate includes Balantidium coli. The sporozoa is preferrably a Plasmodium sp. including P. vivax and P. falciparum, a Toxoplasma gondii, Pneumocystis carinii, Isospora hominis, Cryptosporidium sp. including C. parvum and C. muris.
The nematoda is preferrably a whipworm, a hookworm, a pinworm, an ascarid or a filariodea but not limited thereto.
The whipworm is preferrably Trichuris trichiura or Trichocephalus trichiuris but not limited thereto. However, any other whipworms infecting animals such as dogs, cats and pigs may be included.
The hookworm is preferrably Ancylostoma duodenale or Necator americanus but not limited thereto.
The pinworm is preferrably Enterobius vermicularis or Enterobius gregorii. The ascarid is preferrably A. suum which typically infects pigs or A. lumbricoides which infects humans.
The filariodea is preferrably Wuchereria bancrofti, Onchocerca volvulus, Loa loa or Dirofilaria immitis.
The trematoda is preferably Digenea, but not limited thereto. The Digenea includes Schistosome or non-Schistosome.
The schistosome includes Schistosoma mansoni, Schistosoma haematobium, Schistosoma japonicun and Schistosoma intercalatum, but not limited thereto.
The non-Schistosome includes Fasciolopsis buski, Heterophyes heterophyes, Metagonimus yokogawaii, Gastrodiscoides hominis, Clonorchis sinensis, Fasciola hepatica and Paragonimus westermani, but not limited thereto. The cestoda is preferably Taeniidae or Diphyllobothriidae but not limited thereto.
The Taeniidae includes Taenia solium and Taenia saginata. The Diphyllobothriidae includes Diphyllobothrium sp. such as Diphyllobothrium latum, Diphyllobothrium dendriticum, D. nihonkaiense, D. pacificum, D. cordatum, D. ursi, D. lanceolatum, D. dalliae, and D. yonagoensis. The "parasite antigen" means a molecule derived from a parasite which is capable of inducing humoral response in a host. The parasite antigen can be a surface glyco- protein or a carbohydrate molecule thereof or a lipid molecule.
With respect to particular parasite antigens, the art discloses various parasite antigens. Particularly, WO9526402A1 discloses a helminth parasite antigen characterized by
(i) in native form being an integral membrane protein; (ii) having a native localization in the parasite gut; (iii) being capable of binding to a thiol affinity medium; and (iv) being recognised by sera from immunised animal hosts. WO951267 IAl discloses a helminth parasite antigen possesing aminopeptidase-like activity. US6399077 discloses a noninfectious soluble fraction of a Toxoplasma gondii infected cell culture lysate. US6413521 discloses an isolated and purified antigen conferring protective immunity against a non-obligate blood feeding helminth and which is characterized by possessing aminopeptidase M-like activity. US20070087012A1 discloses a novel Fasciclin Related Adhesive Protein (FRAP) from Plasmodium and related parasites. WO9402169A1 discloses a protective metazoan parasite antigen capable of binding to pepstatin. US4656251 discloses parasite antigens of Dirofilaria immitis. US4839275 circulating parasite antigens of Dirofilaria immitis.
The therapeutic agent against viral or parasite infection can be administered parenterally by hypodermic injection, intravenous injection or intramuscular injection. To prepare the vaccine composition as a formulation for parenteral administration, the vaccine composition of the present invention is prepared as an oil emulsion, which is then formulated as ampoules, syringes or vials. The effective dosage can be determined according to absorption rate, inactivation rate, age, gender, health condition of a patient, and severity of disease, etc.
The present invention also provides a preventive or therapeutic agent for cancer containing the adjuvant and at least one cancer-specific antigen composition as active ingredients. The cancer is preferably a renal cell carcinoma, a melanoma, a chronic lymphocytic leukemia, a lung cancer, a cervical cancer, a stomach cancer, a thyroid cancer, a pancreatic cancer, a breast cancer, a prostate cancer, an ovarian cancer, a cholangioma, a liver cancer, a colon cancer, or a lectal cancer, but not limited thereto.
The "cancer-specific antigen" is a ptotein or an immulogically active fragment thereof which is differentially expressed in cancerous tissues rather than normal tissues. Various cancer-specific antigens are known in the art. For example, gplOO, MART-I and MAGE-I are well-known antigen specific for menanoma. The other cancer-specific antigen includes tyrosinase, CEA (cancer embryonic antigen), PSA (prostate specific antigen), HER2/neu, MAGE-I, MAGE-2, MAGE-3, NY-ESO-I, MUC-I, SART-I or SART-3, TERT (telomerase reverse transcriptase) or partial peptides derived from TERT, WTl or partial peptides derived from WTl, Survivin-2B or partial peptides derived from Survivin-2B, gp75, MDM2, telomerase, alph-1 fetoprotein, CA125, CA15-3, CA19-9, G250 and NY-ESO-I5 but not limited thereto (See WO 2006/078059 and WO 2007/065957).
Additional cancer-associated antigen (CAA) and a method for identifying CAA is disclosed in Miller (Drig Discovery Today, 8: 31-38, 2003) and Kawakami and Rosenberg (Immunol. Res. 16:313, 2003) and Slingluff et a (Curr. Opin. Immunol., 6:733, 1994) and the documents are incoprated by reference.
In addition, the present invention provides use of the adjuvant as a vaccine composition in the manufacture of an immunological therapeutic agent for treating cancer, in that strong cellular immune response is required. In a preferred embodiment of the present invention, the immunological therapeutic agent composition prepared by using the adjuvant of the present invention increased antigen-specific antibody production and IgG2a and IgG2b production (see Figures 1 — 10 and Tables 1 - 4). IgG2a and IgG2b are known to be very effective in anticancer immune response. Therefore, the immunological therapeutic agent composition containing the adjuvant for vaccine of the present invention and an appropriate cancer-specific antigen can be effectively used as a preventive or therapeutic agent for preventing or treating cancer.
The immunological agent for preventing or treating cancer can be administered parenterally by hypodermic injection, intravenous injection or intramuscular injection. To prepare the vaccine composition as a formulation for parenteral administration, the vaccine composition of the present invention is formulated as an oil emulsion, which is then stored as ampoules, syringes, or vials. The effective dosage can be determined according to absorption rate, inactivation rate, age, gender, health condition of a patient, and severity of disease, etc. Mode for Invention
Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples.
However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.
Example 1: Stimulation of immunogenicity of hepatitis B virus (HBV) antigen
Vaccines were prepared with hepatitis B virus antigen and various adjuvants including aluminum hydroxide (Alum; Brenntag Biosector, Germany), Pam3Cys-SKKKK (lipopeptide) (EMC microcollections GmbH, Germany) alone, poly I:C (Sigma, USA) alone, or the mixture of both Pam3Cys-SKKKK and poly I:C, and the induced antibody titer of each vaccine formulation was compared.
<1-1> Preparation of vaccines and administration Vaccines were prepared by mixing hepatitis B virus whole surface antigen (L-HBsAg;
Korean Patent No: 10-0836745) and the said adjuvant, which were administered to mice. In this, L-HBsAg is consisted of S-protein (small protein without pre S 1 and pre S2), M-protein (medium protein with pre S2 only), and L-protein (large protein with both preSl and preS2).
Particularly, as shown in Table 1, 20 μg of Pam3Cys-SKKKK or poly LC or both Pam3Cys-SKKKK and poly LC were mixed with 0.5 μg of L-HBsAg to give a vaccine in oil emulsion form and induced antibody titer by different formulatons was compared. As a positive control, the same amount of antigen was formulated with aluminum hydroxide. Vaccines were injected intra muscularly into 6 weeks old C57BL/6 female mice three times at two weeks intervals. The negative control was injected with PBS without antigen and adjuvant.
Table 1
<l-2> Analysis of immune response
<l-2-l> Antibody titer against HBsAg (S-protein)
Serum was collected before the vaccine administration and 2 weeks after the third vaccine administration and antigen specific antibody generation was analyzed by ELISA to determine antibody titer.
Particularly, a 96-well microplate was coated with recombinant S-protein (Dobeel Corp., Korea) at the concentration of 100 ng/well and blocked by adding 1% BSA for one hour. The microplate was washed and apropriately diluted serum was added to each well and incubated at 37°C for 2 hours. Then, Anti mouse goat IgG-HRP (Horse Radish Peroxidase; Sigma, USA) as a secondary antibody was added to each well and incubated at 370C for one hour. At the end of incubation plates were washed extensively with PBST (PBS with Tween 20), and TMB (3, 3', 5, 5'-tetramethyl benzidine) peroxidase substrate solution (KPL5 USA) was added, and followed by incubation at room temperature for 20 minutes. Then, OD450 was measured with an ELISA reader. Antibody titer was determined as the inverse value of antibody final dilution to give OD reading that is three times high OD of the negative control.
As shown in Table 1 and Figure 1, use of Pam3Cys-SKKKK or poly I: C was more effective than aluminum hydroxide in inducing higher antibody titer against S-protein of HBV envelop protein. Specially, when the mixture of Pam3Cys-SKKKK and poly I:C was used, the induced antibody titer was slightly higher than the combined value of individually induced antibody titer indicating synergistic effect of these two components. The synergistic effect of combined use of Pam3Cys-SKKKK and Poly I:C is more pronounced in the induction of Pre S antibody. This could be due to the amount of antigen used, since the amount of pre S in L-HBsAg preparation is less than 10% of the total. And the 0.5 ug of antigen is near the saturating amount for immune response in mice (data not presented).
<l-2-2> Antibody titer against preS
Antibody titer was determined by the same way as described in Example <l-2-l> except that preS antigen (Dobeel Corp., Korea) was used as an antibody capturing antigen. As shown in Table 1 and Figure 2, the adjuvant mixture containing both
Pam3Cys-SKKKK and poly I:C was more effective in inducing higher antibody titer against preS. Induction of pre S antibody by the adjuvant mixture was synergistic, inducing more than 4 times of pre S antibody compare to the added value of pre S antibody titer induced by Pam3Cys-SKKKK alone and poly I:C alone.
<l-2-3> Isotvpes of induced HBsAg specific antibody
Antibody titer was determined by the same manner as described in Example <l-2-l> except IgGl, IgG2a and IgG2b (mouse monoclonal antibody isotyping reagents; Sigma, USA) were used as a secondary antibody. And IgG2a/IgGl and IgG2b/IgGl ratios were also calculated by using the obtained antibody titer.
As shown in Figure 3 a, isotypes IgG2a and IgG2b were much higher with the adjuvant mixture comparing to the values obtained with aluminum hydroxide. Specially, the induction of IgG2b was more than 20 times of the value obtained with aluminum hydroxide. When the adjuvant mixture comprising Pam3Cys- SKKKK and poly I:C was used, IgG2a/IgGl ratio was about 10 times higher than aluminmum hydroxide. Specially, the production of IgG2b was significantly high, and IgG2b/IgGl ratio was much higher than IgG2a/IgGl (Figure 3b and Figure 3c).
Example 2: Stimulation of immunogenicity of influenza virus antigen
Influenza virus antigen was formulated with aluminum hydroxide, Pam3Cys-SKKKK alone, poly I:C alone, or with the mixture of Pam3Cys-SKKKK and Poly I:C as adjuvants and induced antibody titer was determined using influenza virus antigen as capturing antigen as for HBsAg antibody assay.
<2-l> Formulation of Influenza virus antigen and administration
Different formulations were prepared by mixing recombinant split vaccine antigen (Korea Vaccine Co., Ltd, Korea) and the said adjuvant, which were administered to mice. The antigen was prepared by infecting the allantoic sac of a developing egg with influenza virus strains A/New Caledonia/20/99(H1N1), a/Wisconsin/67/2005(H3N2) and B/Malaysia/2506/2004, culturing, purifying and inactivating thereof.
Particularly, as shown in Table 2, 20 βg of Pam3Cys-SKKKK alone, poly I:C alone, the mixture of Pam3Cys-SKKK and Poly LC, or aluminum hydroxide were mixed with 1.8 βg of the split vaccine antigen to give various different vaccine formulations in oil emulsion form. These different formulations were given by intramuscular injection to 5 week old C57BL/6 female mice two times at three weeks interval. The negative control received only PBS, while the positive control received the said antigen alone without any adjuvant. All group contains 6 mice. Table 2
<2-2> Analysis of immune response
Serum was collected from each mouse before the vaccine administration and 2 weeks after the second vaccine administration and antigen specific antibody generation was analyzed by ELISA to determine antibody titer.
<2-2-l> Determination of HA antibody titer
Antibody titer was determined by the same manner as described in Example <l-2-l> except that HA antigen (Korea Vaccine Co., Ltd, Korea) was used as an antibody capturing antigen.
As shown in Table 2 and Figure 4, the mixture of Pam3Cys-SKKKK and poly LC was more effective in inducing higher antibody titer against HA (synergistic effect) than single component or aluminum hydroxide. <2-2-2> Isotypes of induced HA specific antibody
Antibody titer was determined by the same way as described in Example <2-2-l> except that IgGl and IgG2a were used as secondary antibodies for isotype determination. IgG2a/IgGl and IgG2b/IgGl ratios were also calculated by using the obtained antibody titer. Figure 5 a shows antibody titers of isotypes IgGl, and IgG2a. Isotype ratios were also calculated using the antibody titers. IgG2a/IgGl value was specifically high (Figure 5b) with the adjuvant mixture compare to the value obtained with single components or Aluminum hydroxide. Example 3: Preparation of a powerful vaccine using recombinant HBsAg (S-protein from Hansenula polymorphά) and recombinant preS protein (from Saccharomyces cerevisiae)
Various vaccine formulations were prepared with recombinant HBsAg and recombinant preS protein by using aluminum hydroxide or the mixture of Pam3Cys-SKKKK and poly I:C as adjuvants, and immune reponses were compared.
<3-l> Preparation of vaccines and administration thereof
Vaccines were prepared by mixing recombinant HBsAg (Dobeel Corp., Korea), recombinant preS protein (Dobeel Corp., Korea) and said adjuvants, and they were administered by intra-muscular injection to mice. The recombinant HBsAg contained only S-protein without preS antigen and the recombinant preS protein prepared as a particle type by conjugating them to colloidal gold were used as antigens.
Particularly, a mixture containing 20 βg each of Pam3Cys-SKKKK and poly I:C was used as adjuvant with 0.5 βg of recombinant S-protein and 5 βg of preS protein to give a vaccine in oil emulsion form. Each test vaccine contained 0.5 βg of the recombinant S-protein and 5 βg of the preS per dose, which was then given by intramuscular injection to 5 week old C57BL/6 female mice three times at two weeks intervals.
Negative control is the group received PBS only. Positive control is the group received the mixture comprising aluminum hydroxide, S-protein, and colloidal gold conjugated recombinant preS antigen. Experimental group 1 is the group received vaccine prepared by mixing emulsified S-protein, Pam3Cys-SKKKK and poly I:C and colloidal gold conjugated recombinant preS antigen. Experimental group 2 is the group received vaccine prepared by emulsification of all components together, including S-protein, colloidal gold conjugated recombinant preS antigen and the mixture of Pam3Cys-SKKKK and poly LC. <3-2> Analysis of immune responses
Serum samples were collected before the vaccine administration and 2 weeks after the third vaccine administration, and antigen specific antibody generation was analyzed by ELISA and expressed as antibody titer. <3-2-l> Antibody titer against S-protein
Antibody titer against S-protein was determined by the same way as described in Example <l-2-l>.
As shown in Table 3 and Figure 6, the adjuvant mixture of Pam3Cys-SKKKK and poly LC induced more than 10 times higher antibody titer against S-protein than aluminum hydroxide. Particularly, the vaccine prepared by emulsifying all components together induced higher antibody titer than the vaccine prepared by emulsifying S-protein with the said adjuvant first, and adding the conjugated pre S.
Table 3
<3-2-2> Pre S antibody titer
Pre S antibody titer was determined by the same manner as described in Example <l-2-2>.
As shown in Table 3 and Figure 7, use of Pam3Cys-SKKKK and poly I:C as adjuvant mixture induced higher antibody titer against pre S compared to the use of aluminum hydroxide. Particularly, the vaccine prepared by emulsifying all components together — including HBsAg and preS antigen with the said adjuvant — induced pre S antibody most efficiently, inducing more than 3 times of the antibody by aluminum hydroxide.
<3-2-3> Isotypes of induced HBsAg specific antibody
Isotype antibody titer was determined by the same method as described in Example <l-2-3>. And IgG2a/IgGl and IgG2b/IgGl ratios were calculated from the obtained antibody titer.
As shown in Figure 8a, antibody titers of isotypes IgGl, IgG2a and IgG2b were obtained. Isotype ratios were also calculated using the antibody titers. When the adjuvant mixture of Pam3Cys-SKKKK and poly I:C was used, IgG2a/IgGl and IgG2b/IgGl ratios were higher comparing to aluminum hydroxide. In particular, the vaccine, prepared by mixing HBsAg and preS antigen with the said adjuvant and emulsifying together, was confirmed more effective than the vaccine prepared by mixing preS antigen with emulsified HBsAg and the said adjuvant (Figures 8b and 8c).
Example 4: Adjuvant composition using various lipopeptides
To test the synergistic adjuvant effect of various lipopeptides with poly LC, Pam3Cys-SKKKK, Pam3Cys-SR8, or FSL-I (Fibroblast-stimulating lipopeptide) is formulated with hepatitis B virus antigen.
<4-l> Preparation of vaccines and administration
Vaccines were prepared by mixing hepatitis B virus whole surface antigen that was preadsorbed on aluminum hydroxide and said adjuvant components, and they were administered to mice. In the course of experiments, it was noticed that the aluminum hydroxide adsorbed antigen is more stable, and the aluminum hydroxide has no noticeable effect on the adjuvant effect of lipopeptide and poly LC mixture (data not presented).
Particularly, as shown in Table 4, 0.5 μg of L-HBsAg absorbed to aluminum hydroxide was formulated with 20 βg of each of the lipopeptides or poly LC (experimental group 1 -4). Also the same amount of antigen was formulated with the mixture of lipopeptide and poly LC (experimental group 5-7).
Vaccines were injected intra muscularly into 6 weeks old C57BL/6 female mice three times at two weeks intervals. The negative control was administered only with PBS without the vaccine and the antigen, while the positive control was administered with the said antigen formulated with aluminum hydroxide. Table 4
<4-2> Analysis of immune response Serum was collected from each mouse before the vaccine administration and 2 weeks after the last vaccine administration and antigen specific antibody generation was analyzed by ELISA to determine antibody titer.
<4-2-l> Antibody titer against HBsAg (S-protein) Antibody titer was determined by the same method as described in Example <l-2-l>.
As shown in Table 4 and Figure 9, when the mixture of the lipopeptide (such as
Pam3Cys-SKKKK and Pam3Cys-SR8) and poly LC was used, the induced antibody titer was slightly higher than the combined value of individually induced antibody titer indicating synergistic effect of these two components.
<4-2-2> Antibody titer against υreS
Antibody titer was determined by the same way as described in Example <l-2-l> except that preS antigen (Dobeel Corp., Korea) was used as an antibody capturing antigen.
As shown 1 Table 4 and Figure 10, the adjuvant mixture containing one of the lipopeptides and poly LC was more effective in inducing higher antibody titer against preS.
The synergistic effect of combined use of lipopeptides other than Pam3Cys-SKKKK and poly
LC is also more dramatic for pre S antibody generation as seen before with Pam3Cys-SKKKK and poly LC in the example <l-2-2>. <4-2-3> Isotypes of induced HBsAg specific antibody
Antibody titer was determined by the same manner as described in Example <l-2-l> except IgGl5 IgG2a and IgG2b were used as a secondary antibody. And IgG2a/IgGl and IgG2b/IgGl ratios were also calculated by using the obtained antibody titer.
As shown in Figure 11a, antibody titers of isotypes IgG2a and IgG2b were much higher when the combination of the lipopeptide and poly I:C was used as an adjuvant compared to the value induced with aluminum hydroxide as adjuvant. Consequently the IgG2a/IgGl and IgG2b/IgGl ratio were also higher when the adjuvant mixture was used (Figure l ib, c). The synergistic effects were similar when the Pam3Cys-SKKKK, Pam3Cys-SR8 or FLS-I was used as the lipopeptide component. Specially the combination of Pam3Cys-SKKKK and poly I:C was the most effective.
Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.

Claims

1. An adjuvant for a vaccine comprising one or more lipopeptide and poly I:C(polyinosinic:polycytidylic acid) in that each components are synergistic in stimulating induction of adaptive immune responses.
2. The adjuvant according to claim 1, wherein the lipopeptide is selected from the group consisting of Pam3Cys-SKKKK, Pam3Cys-SR8, FLS-I, PHC-SKKKK, Ole2PamCys-SKKKK, Pam2Cys-SKKKK, PamCys(Pam)-SKKKK, Ole2Cys-SKKKK, Myr2Cys-SKKKK, PamDhc-SKKKK, PamCSKKKK and Dhc-SKKKK.
3. The adjuvant according to claim 1, wherein the poly I:C is 50 - 2,000 bp in length.
4. A vaccine composition comprising the adjuvant of claim 1 and at least one antigen.
5. The vaccine composition according to claim 4, wherein the antigen is selected as single or multiple component from the group consisting of a protein of a pathogen, a recombinant protein, a peptide, a happen, a polysaccharide, a glycoprotein, a lipopolysaccharide, a DNA molecule (polynucleotide), a cancer cell, and a micro-organism.
6. The vaccine composition according to claim 4, wherein the antigen is selected from the group consisting of L-HBsAg, influenza HA, S-protein, and preS.
7. The vaccine composition according to claim 4, which is capable of efficiently inducing cell mediated immunity and producing appropriate antigen-specific high quality antiboies, indcluding IgGl, IgG2a and IgG2b.
8. The vaccine composition according to claim 7, wherein the appropriate high quality antibodies are IgGl, IgG2a and IgG2b types.
9. A method for generating appropriate high quality antibody comprising steps of administrating the vaccine composition of claim 4 to a subject in need thereof.
10. The method according to claim 9, wherein the method facilitates mass-production of an appropriate high quality antibody.
11. A method for enhancing ThI immune response comprising administrating the vaccine composition of claim 4 to a subject in need thereof.
12. A therapeutic agent for viral or parasitic infection comprising the adjuvant of claim 1 and at least one viral antigen or parasite antigen.
13. The therapeutic agent according to claim 12, wherein the viral antigen is influenza virus antigen (HA: haemagglutinin or neuraminidase antigen), human papilloma virus (HPV) antigen, human immunodeficiency virus (HIV) antigen (GP- 120, GP- 160), vesicular stomatitis virus antigen (vesicular stomatitis virus glycoprotein), cytomegalovirus (CMV) antigen, hepatitis antigen [hepatitis A(HAV), B(HBV), C(HCV), D(HDV) and G(HGV): L-HBsAg, S-HBsAg, M-HBsAg, pre S], respiratory synctytial virus (RSV) antigen or herpes simplex virus antigen.
14. The therapeutic agent according to claim 12, wherein the parasite is protozoa, nematoda, trematoda or cestoda.
15. A preventive or therapeutic vaccine for cancer comprising the adjuvant of claim 1 and at least one cancer-specific antigen.
16. The preventive or therapeutic vaccine according to claim 15, wherein the cancer is renal cell carcinoma, a melanoma, a chronic lymphocytic leukemia, a lung cancer, a cervical cancer, a stomach cancer, a thyroid cancer, a pancreatic cancer, a breast cancer, a prostate cancer, an ovarian cancer, a cholangioma, a liver cancer, a colon cancer, or a lectal cancer.
17. The preventive or therapeutic vaccine for cancer according to claim 15, wherein the cancer-specific antigen is gplOO, MART-I and MAGE-I, tyrosinase, CEA (cancer embryonic antigen), PSA (prostate specific antigen), HER2/neu, MAGE-I, MAGE-2, MAGE-3, NY-ESO-I, MUC-I, SART-I or SART-3, TERT (telomerase reverse transcriptase) or a partial peptide derived from TERT, WTl or a partial peptide derived from WTl, Survivin-2B or a partial peptide derived from Survivin-2B, gp75, MDM2, telomerase, alρh-1 fetoprotein, CA125, CA15-3, CA19-9, G250 or NY-ESO-I.
18. Use of the adjuvant of claim 1 and at least one viral or parasite antigen in the manufacture of a therapeutic agent for treating viral or parasite infection.
19. Use of the adjuvant of claim 1 and at least one cancer-specific antigen in the manufacture of an immunological agent for preventing or treating cancer.
EP08856104.8A 2007-12-07 2008-11-20 A powerful vaccine composition comprising a lipopeptide and poly i:c as an adjuvant Active EP2214705B1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR1020070126775A KR100900837B1 (en) 2007-12-07 2007-12-07 A powerful vaccine composition comprising lipopeptide and poly(i:c) as an adjuvant
PCT/KR2008/006842 WO2009072767A2 (en) 2007-12-07 2008-11-20 A powerful vaccine composition comprising a lipopeptide and poly i:c as an adjuvant

Publications (3)

Publication Number Publication Date
EP2214705A2 true EP2214705A2 (en) 2010-08-11
EP2214705A4 EP2214705A4 (en) 2011-11-30
EP2214705B1 EP2214705B1 (en) 2014-08-20

Family

ID=40718329

Family Applications (1)

Application Number Title Priority Date Filing Date
EP08856104.8A Active EP2214705B1 (en) 2007-12-07 2008-11-20 A powerful vaccine composition comprising a lipopeptide and poly i:c as an adjuvant

Country Status (9)

Country Link
US (1) US8216595B2 (en)
EP (1) EP2214705B1 (en)
JP (1) JP5485165B2 (en)
KR (1) KR100900837B1 (en)
CN (1) CN101883585B (en)
AU (1) AU2008332196B2 (en)
CA (1) CA2706101C (en)
HK (1) HK1149710A1 (en)
WO (1) WO2009072767A2 (en)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3281641A4 (en) * 2015-04-07 2018-12-19 Cytlimic Inc. Medicine
US10526388B2 (en) 2015-03-09 2020-01-07 Cytlimic Inc. Peptide derived from GPC3, pharmaceutical composition for treatment or prevention of cancer using the same, immunity inducer, and method for producing antigen-presenting cells
US10537626B2 (en) 2014-10-07 2020-01-21 Cytlimic Inc. HSP70-derived peptide, pharmaceutical composition for treating or preventing cancer using same, immunity inducer, and method for producing antigen presenting cell
US11291718B2 (en) 2016-10-11 2022-04-05 Cytlimic Inc. Method for treating cancer by administering a toll-like receptor agonist and LAG-3 IgG fusion protein

Families Citing this family (47)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080299140A1 (en) * 2002-05-24 2008-12-04 The Regents Of The University Of California, Immunogenic Composition and Peptide Sequences for Prevention and Treatment of an Hsv Condition
ES2531142T3 (en) 2005-10-17 2015-03-11 Sloan Kettering Inst Cancer Class II HLA binding WT1 peptides, and compositions and methods comprising them
US8067237B2 (en) 2005-12-13 2011-11-29 President And Fellows Of Harvard College Scaffolds for cell transplantation
PL2010209T3 (en) 2006-04-10 2017-04-28 Sloan Kettering Inst For Cancer Res Immunogenic wt-1 peptides and methods of use thereof
US9770535B2 (en) 2007-06-21 2017-09-26 President And Fellows Of Harvard College Scaffolds for cell collection or elimination
US8466259B2 (en) 2007-12-07 2013-06-18 National Health Research Institutes Adjuvants
US9370558B2 (en) 2008-02-13 2016-06-21 President And Fellows Of Harvard College Controlled delivery of TLR agonists in structural polymeric devices
CA2715460C (en) 2008-02-13 2020-02-18 President And Fellows Of Harvard College Continuous cell programming devices
US8287880B2 (en) 2009-06-02 2012-10-16 National Health Research Institutes Lipidated vaccine against dengue virus infection
WO2010148496A1 (en) 2009-06-22 2010-12-29 National Health Research Institutes Lipidated tumor- associated antigens and immunotherapeutic compositions
WO2011014871A1 (en) 2009-07-31 2011-02-03 President And Fellows Of Harvard College Programming of cells for tolerogenic therapies
KR102157971B1 (en) 2010-10-06 2020-09-18 프레지던트 앤드 펠로우즈 오브 하바드 칼리지 Injectable, pore-forming hydrogels for materials-based cell therapies
TWI507413B (en) 2010-11-15 2015-11-11 Nat Health Research Institutes Lipidated polyepitope vaccines
TW201221642A (en) 2010-11-15 2012-06-01 Nat Health Research Institutes Method of producing lipidated polypeptides
WO2012139094A2 (en) 2011-04-08 2012-10-11 H. Lee Moffitt Cancer Center And Research Institute, Inc. Method of developing a vaccine using peptide-poly ic complexes
US9675561B2 (en) 2011-04-28 2017-06-13 President And Fellows Of Harvard College Injectable cryogel vaccine devices and methods of use thereof
US10045947B2 (en) 2011-04-28 2018-08-14 President And Fellows Of Harvard College Injectable preformed macroscopic 3-dimensional scaffolds for minimally invasive administration
CA2838125A1 (en) 2011-06-03 2012-12-06 President And Fellows Of Harvard College In situ antigen-generating cancer vaccine
NL2007536C2 (en) * 2011-10-05 2013-04-08 Academisch Ziekenhuis Leiden Lumc Adjuvant compound.
ES2855474T3 (en) 2011-10-06 2021-09-23 Immunovaccine Technologies Inc Liposome compositions comprising an adjuvant that activates or increases TLR2 activity and uses thereof
US20150104413A1 (en) 2012-01-13 2015-04-16 Memorial Sloan Kettering Cancer Center Immunogenic wt-1 peptides and methods of use thereof
ES2773895T3 (en) 2012-04-16 2020-07-15 Harvard College Mesoporous Silica Compositions to Modulate Immune Responses
CN102949717A (en) * 2012-07-06 2013-03-06 中国疾病预防控制中心病毒病预防控制所 Novel hepatitis B vaccine preparation containing poly I:C adjuvant
WO2014047533A1 (en) * 2012-09-21 2014-03-27 Frank Bedu-Addo Improved vaccine compositions and methods of use
KR101669142B1 (en) 2012-10-10 2016-10-25 연세대학교 산학협력단 A novel adjuvant of vaccine for eye-drop vaccination
US10815273B2 (en) 2013-01-15 2020-10-27 Memorial Sloan Kettering Cancer Center Immunogenic WT-1 peptides and methods of use thereof
LT2945647T (en) 2013-01-15 2021-02-25 Memorial Sloan Kettering Cancer Center Immunogenic wt-1 peptides and methods of use thereof
KR101501583B1 (en) * 2013-03-29 2015-03-12 주식회사 차백신연구소 An adjuvant comprising lipopeptide and poly(I:C), and improved form of vaccine composition using thereof
EP3137105A4 (en) 2014-04-30 2017-12-27 President and Fellows of Harvard College Combination vaccine devices and methods of killing cancer cells
CN104208670B (en) * 2014-09-09 2017-07-28 韩自勤 A kind of pharmaceutical composition for treating hepatitis B
EP3242883A4 (en) 2015-01-06 2018-10-17 ImmunoVaccine Technologies Inc. Lipid a mimics, methods of preparation, and uses thereof
US11786457B2 (en) 2015-01-30 2023-10-17 President And Fellows Of Harvard College Peritumoral and intratumoral materials for cancer therapy
CN114099793A (en) 2015-04-10 2022-03-01 哈佛学院院长等 Immune cell capture device and methods of making and using same
AU2016355926A1 (en) 2015-11-18 2018-05-31 Immunovaccine Technologies Inc. Adjuvanting systems and water-free vaccine compositions comprising a polyi:C polynucleotide adjuvant and a lipid-based adjuvant
CN115531609A (en) 2016-02-06 2022-12-30 哈佛学院校长同事会 Remodeling hematopoietic niches to reconstitute immunity
CN115305229A (en) 2016-07-13 2022-11-08 哈佛学院院长等 Antigen presenting cell mimetic scaffolds and methods of making and using same
CN107261136B (en) * 2017-07-31 2020-07-28 中国医学科学院医学生物学研究所 Application of sodium polyphosphate and vaccine containing sodium polyphosphate
KR102098097B1 (en) 2017-08-16 2020-05-26 주식회사 차백신연구소 Vaccine adjuvant comprising lipopeptide-inserted liposome as an effective ingredient and using thereof
CA3075526A1 (en) * 2017-09-12 2019-03-21 Garvan Institute Of Medical Research Methods for reducing or shutting down lactation in non-human mammals and reagents therefor
CN109793891A (en) * 2017-11-17 2019-05-24 韩震 Application of the polypeptide compound in the adjuvant that preparation improves therapeutic antibodies curative effect and composition and preparation method thereof
CN109200056A (en) * 2018-11-20 2019-01-15 苏州系统医学研究所 Purposes of the poly IC in prevention and treatment pancreatitis and its associated disease
WO2021231712A1 (en) * 2020-05-15 2021-11-18 Rxcell Inc. Hypoimmunogenic cells and uses thereof in immune responses
US20230390384A1 (en) * 2020-11-09 2023-12-07 Cha Vaccine Research Institute Co., Ltd Recombinant covid-19 vaccine composition comprising lipopeptide and poly (i:c) adjuvant, and use thereof
KR102292147B1 (en) * 2020-11-18 2021-08-23 주식회사 차백신연구소 Pharmaceutical composition, pharmaceutical combination preparation, combination preparation kit for the prevention or treatment of chronic hepatitis B comprising an oral antiviral agents and a therapeutic vaccine comprising lipopeptide and poly(I:C) adjuvant as an active ingredients
WO2022250416A1 (en) 2021-05-24 2022-12-01 주식회사 차백신연구소 Immuno-oncology therapeutic composition using adjuvant including lipopeptides and poly (i:c)
KR102375057B1 (en) * 2021-09-29 2022-03-17 주식회사 애스톤사이언스 Anti-cancer vaccine composition comprising HSP90 antigenic peptide
WO2024149888A1 (en) 2023-01-12 2024-07-18 Isa Pharmaceuticals B.V. Improved lipopeptide quantification

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006040076A2 (en) * 2004-10-08 2006-04-20 Johannes-Gutenberg- Universität Mainz Vaccination adjuvants pam3cys, poly(i:c), imiquimod, loxoribine, r-848 and cpg-dna together with mhc i or mhc ii epitopes

Family Cites Families (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4839275A (en) 1983-12-01 1989-06-13 The Jewish Hospital Circulating antigens of dirofilaria immitis, monoclonal antibodies specific therefor and methods of preparing such antibodies and detecting such antigens
US4656251A (en) 1985-01-28 1987-04-07 Mallinckrodt, Inc. Rapid separation of Dirofilaria immitis immune complexes
ZW8893A1 (en) 1992-07-21 1994-06-01 Pitman Moore Inc Vaccines
GB9322702D0 (en) 1993-11-03 1993-12-22 Agricultural & Food Res Vaccines
AU701642B2 (en) 1994-03-25 1999-02-04 Mallinckrodt Veterinary, Inc. Vaccines against helminthic parasites
WO1998006434A1 (en) 1996-08-09 1998-02-19 Trustees Of Dartmouth College METHOD OF AUGMENTING T CELL-MEDIATED IMMUNITY AGAINST $i(TOXOPLASMA GONDII)
AU2002327338A1 (en) * 2001-07-25 2003-02-17 New York University Use of glycosylceramides as adjuvants for vaccines against infections and cancer
EP1578419A4 (en) * 2002-12-30 2008-11-12 3M Innovative Properties Co Immunostimulatory combinations
GB0321615D0 (en) * 2003-09-15 2003-10-15 Glaxo Group Ltd Improvements in vaccination
AU2003290460A1 (en) * 2003-12-24 2005-07-14 Leiden University Medical Center Synthetic protein as tumor-specific vaccine
CN101111258B (en) 2005-01-19 2011-07-06 大日本住友制药株式会社 Emulsified composition for dilution and cancer vaccine composition
CA2603356A1 (en) * 2005-03-31 2006-10-05 Leiden University Medical Center Methods and means for diagnostics, prevention and treatment of mycobacterium infections and tuberculosis disease
ES2380481T3 (en) * 2005-06-08 2012-05-14 Yisheng Biopharma (Singapore) Pte. Ltd. Immunogenic substances comprising an adjuvant based on polyinosinic acid and polycycidyl acid
JP4828189B2 (en) * 2005-09-14 2011-11-30 雅美 森山 Secretory IgA and IgG antibody inducer
US7438916B2 (en) 2005-10-14 2008-10-21 Virginia Tech Intellectual Properties, Inc. Therapeutic target for protozoal diseases
CA2632797A1 (en) * 2005-12-01 2007-06-07 The Provost, Fellows And Scholars Of The College Of The Holy And Undivid Ed Trinity Of Queen Elizabeth Near Dublin Compositions and methods relating to treatment of cancer and infectious diseases using toll like receptor agonists
EP1795599A1 (en) 2005-12-09 2007-06-13 Schuler, Gerold, Prof. Dr. Methods for generating antigen-specific effector T cells
KR100836745B1 (en) 2007-01-31 2008-06-10 (주)두비엘 An hbv vaccine and a process of preparing the same

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006040076A2 (en) * 2004-10-08 2006-04-20 Johannes-Gutenberg- Universität Mainz Vaccination adjuvants pam3cys, poly(i:c), imiquimod, loxoribine, r-848 and cpg-dna together with mhc i or mhc ii epitopes

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
ASAHI-OZAKI Y ET AL: "Intranasal administration of adjuvant-combined recombinant influenza virus HA vaccine protects mice from the lethal H5N1 virus infection", MICROBES AND INFECTION, ELSEVIER, PARIS, FR, vol. 8, no. 12-13, 1 October 2006 (2006-10-01), pages 2706-2714, XP025243408, ISSN: 1286-4579, DOI: 10.1016/J.MICINF.2006.07.018 [retrieved on 2006-10-01] *
BAGCHI ARANYA ET AL: "MyD88-dependent and MyD88-independent pathways in synergy, priming, and tolerance between TLR agonists.", JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950) 15 JAN 2007 LNKD- PUBMED:17202381, vol. 178, no. 2, 15 January 2007 (2007-01-15), pages 1164-1171, XP000002658393, ISSN: 0022-1767 *
LAHIRI A ET AL: "Engagement of TLR signaling as adjuvant: Towards smarter vaccine and beyond", VACCINE, ELSEVIER LTD, GB, vol. 26, no. 52, 9 December 2008 (2008-12-09), pages 6777-6783, XP025711304, ISSN: 0264-410X, DOI: 10.1016/J.VACCINE.2008.09.045 [retrieved on 2008-11-27] *
LUHRMANN ET AL: "Improved intranasal immunization with live-attenuated measles virus after co-inoculation of the lipopeptide MALP-2", VACCINE, ELSEVIER LTD, GB, vol. 23, no. 39, 15 September 2005 (2005-09-15), pages 4721-4726, XP005040629, ISSN: 0264-410X, DOI: 10.1016/J.VACCINE.2005.05.009 *
See also references of WO2009072767A2 *
WARGER TOBIAS ET AL: "Synergistic activation of dendritic cells by combined Toll-like receptor ligation induces superior CTL responses in vivo.", BLOOD 15 JUL 2006 LNKD- PUBMED:16537810, vol. 108, no. 2, 15 July 2006 (2006-07-15) , pages 544-550, XP000002658391, ISSN: 0006-4971 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10537626B2 (en) 2014-10-07 2020-01-21 Cytlimic Inc. HSP70-derived peptide, pharmaceutical composition for treating or preventing cancer using same, immunity inducer, and method for producing antigen presenting cell
US11304997B2 (en) 2014-10-07 2022-04-19 Cytlimic Inc. HSP70-derived peptide, pharmaceutical composition for treating or preventing cancer using same, immunity inducer, and method for producing antigen-presenting cell
US10526388B2 (en) 2015-03-09 2020-01-07 Cytlimic Inc. Peptide derived from GPC3, pharmaceutical composition for treatment or prevention of cancer using the same, immunity inducer, and method for producing antigen-presenting cells
US10875900B2 (en) 2015-03-09 2020-12-29 Cytlimic Inc. Peptide derived from GPC3, pharmaceutical composition for treatment or prevention of cancer using same, immunity inducer, and method for producing antigen-presenting cells
EP3281641A4 (en) * 2015-04-07 2018-12-19 Cytlimic Inc. Medicine
US10842848B2 (en) 2015-04-07 2020-11-24 Cytlimic Inc. Method for treating cancer by adminstering poly I:C and LAG-3-IgG fusion protein
US11491204B2 (en) 2015-04-07 2022-11-08 Cytlimic Inc. Composition comprising poly I:C and LAG-3-IGG fusion protein
US11291718B2 (en) 2016-10-11 2022-04-05 Cytlimic Inc. Method for treating cancer by administering a toll-like receptor agonist and LAG-3 IgG fusion protein
US11759518B2 (en) 2016-10-11 2023-09-19 Nec Corporation Medicine for treating cancer by administering a toll-like receptor agonist and LAG-3 IgG fusion protein

Also Published As

Publication number Publication date
KR100900837B1 (en) 2009-06-04
CA2706101A1 (en) 2009-06-11
CN101883585B (en) 2013-04-17
WO2009072767A3 (en) 2009-07-23
HK1149710A1 (en) 2011-10-14
EP2214705B1 (en) 2014-08-20
US8216595B2 (en) 2012-07-10
JP5485165B2 (en) 2014-05-07
EP2214705A4 (en) 2011-11-30
JP2011506309A (en) 2011-03-03
CN101883585A (en) 2010-11-10
WO2009072767A2 (en) 2009-06-11
AU2008332196B2 (en) 2011-09-15
US20090155308A1 (en) 2009-06-18
AU2008332196A1 (en) 2009-06-11
CA2706101C (en) 2014-06-03

Similar Documents

Publication Publication Date Title
AU2008332196B2 (en) A powerful vaccine composition comprising a lipopeptide and poly I:C as an adjuvant
RU2121849C1 (en) Vaccine composition against hepatitis, a method of hepatitis prophylaxis
KR101501583B1 (en) An adjuvant comprising lipopeptide and poly(I:C), and improved form of vaccine composition using thereof
US11759517B2 (en) Pharmaceutical composition that includes the surface and nucleocapsid antigens of the hepatitis B virus
AU2018233208B2 (en) Immunopotentiator, immunotherapeutic pharmaceutical composition and its preparation and use
WO2018166298A1 (en) Immunopotentiator, immunotherapeutic pharmaceutical composition and its preparation and use
AU2002309245B2 (en) Vaccines including as an adjuvant type 1 IFN and processes related thereto
KR102292147B1 (en) Pharmaceutical composition, pharmaceutical combination preparation, combination preparation kit for the prevention or treatment of chronic hepatitis B comprising an oral antiviral agents and a therapeutic vaccine comprising lipopeptide and poly(I:C) adjuvant as an active ingredients
AU2002309245A1 (en) Vaccines including as an adjuvant type 1 IFN and processes related thereto
WO2021045073A1 (en) Seasonal influenza vaccine capable of inducing virus-specific antibody into nasal cavity
US20230390384A1 (en) Recombinant covid-19 vaccine composition comprising lipopeptide and poly (i:c) adjuvant, and use thereof
WO2021172418A1 (en) Method for producing inactivated influenza vaccine and vaccine composition thereof
Park et al. Immune responses of mice to influenza subunit vaccine in combination with CIA07 as an adjuvant
QU et al. Effect of Plasmid DNA on Immunogenicity of HBsAg–Anti-HBs Complex
WO2024191777A2 (en) Influenza virus immunogenic composition and method using the same
CN1882359A (en) Pharmaceutical compositions for therapeutic use
Madaan Heplisav V
JPH059130A (en) Vaccine preparation containing immunoglobulin
Fastier et al. An adjuvant vaccine against infectious canine hepatitis

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20100521

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MT NL NO PL PT RO SE SI SK TR

AX Request for extension of the european patent

Extension state: AL BA MK RS

DAX Request for extension of the european patent (deleted)
RIC1 Information provided on ipc code assigned before grant

Ipc: A61K 39/12 20060101AFI20111020BHEP

Ipc: A61K 39/145 20060101ALI20111020BHEP

Ipc: A61K 39/29 20060101ALI20111020BHEP

Ipc: A61K 39/385 20060101ALI20111020BHEP

Ipc: C07K 16/08 20060101ALI20111020BHEP

Ipc: A61K 39/39 20060101ALI20111020BHEP

A4 Supplementary search report drawn up and despatched

Effective date: 20111027

17Q First examination report despatched

Effective date: 20120822

REG Reference to a national code

Ref country code: DE

Ref legal event code: R079

Ref document number: 602008034027

Country of ref document: DE

Free format text: PREVIOUS MAIN CLASS: A61K0039385000

Ipc: A61K0039120000

RIC1 Information provided on ipc code assigned before grant

Ipc: A61K 39/29 20060101ALI20140130BHEP

Ipc: A61K 39/145 20060101ALI20140130BHEP

Ipc: C07K 16/08 20060101ALI20140130BHEP

Ipc: A61K 39/12 20060101AFI20140130BHEP

Ipc: A61K 39/385 20060101ALI20140130BHEP

Ipc: A61K 39/39 20060101ALI20140130BHEP

Ipc: A61K 39/00 20060101ALI20140130BHEP

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

INTG Intention to grant announced

Effective date: 20140313

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MT NL NO PL PT RO SE SI SK TR

REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: CH

Ref legal event code: EP

REG Reference to a national code

Ref country code: AT

Ref legal event code: REF

Ref document number: 683060

Country of ref document: AT

Kind code of ref document: T

Effective date: 20140915

REG Reference to a national code

Ref country code: IE

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: CH

Ref legal event code: NV

Representative=s name: OFFICE ERNEST T. FREYLINGER S.A., CH

REG Reference to a national code

Ref country code: DE

Ref legal event code: R096

Ref document number: 602008034027

Country of ref document: DE

Effective date: 20141002

REG Reference to a national code

Ref country code: NL

Ref legal event code: T3

REG Reference to a national code

Ref country code: AT

Ref legal event code: MK05

Ref document number: 683060

Country of ref document: AT

Kind code of ref document: T

Effective date: 20140820

REG Reference to a national code

Ref country code: LT

Ref legal event code: MG4D

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20140820

Ref country code: BG

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20141120

Ref country code: ES

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20140820

Ref country code: GR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20141121

Ref country code: FI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20140820

Ref country code: LT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20140820

Ref country code: PT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20141222

Ref country code: NO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20141120

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LV

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20140820

Ref country code: IS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20141220

Ref country code: AT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20140820

Ref country code: HR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20140820

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: EE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20140820

Ref country code: SK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20140820

Ref country code: RO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20140820

Ref country code: CZ

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20140820

Ref country code: DK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20140820

REG Reference to a national code

Ref country code: DE

Ref legal event code: R097

Ref document number: 602008034027

Country of ref document: DE

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: PL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20140820

PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MC

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20140820

Ref country code: BE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20141130

Ref country code: LU

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20141120

26N No opposition filed

Effective date: 20150521

REG Reference to a national code

Ref country code: IE

Ref legal event code: MM4A

REG Reference to a national code

Ref country code: FR

Ref legal event code: PLFP

Year of fee payment: 8

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20141120

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20140820

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: CY

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20140820

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: HU

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT; INVALID AB INITIO

Effective date: 20081120

Ref country code: MT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20140820

Ref country code: BE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20140820

Ref country code: TR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20140820

REG Reference to a national code

Ref country code: FR

Ref legal event code: PLFP

Year of fee payment: 9

REG Reference to a national code

Ref country code: FR

Ref legal event code: PLFP

Year of fee payment: 10

REG Reference to a national code

Ref country code: FR

Ref legal event code: PLFP

Year of fee payment: 11

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: NL

Payment date: 20231026

Year of fee payment: 16

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: GB

Payment date: 20231019

Year of fee payment: 16

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: IT

Payment date: 20231026

Year of fee payment: 16

Ref country code: FR

Payment date: 20231024

Year of fee payment: 16

Ref country code: DE

Payment date: 20231017

Year of fee payment: 16

Ref country code: CH

Payment date: 20231201

Year of fee payment: 16